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Enzymatic probing of chromatin conformation using the comet assay Thompson, Jennifer Jane 1997

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ENZYMATIC PROBING OF CHROMATIN CONFORMATION USING THE COMET ASSAY  by JENNIFER J A N E T H O M P S O N B.Sc. York University, 1995  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Physics and Astronomy  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A July, 1997 © Jennifer Jane Thompson, 1997  In  presenting  this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  11  ABSTRACT  Chromatin structure is thought to play an important role in the response of mammalian cells to ionizing radiation.  Structural differences amongst cell types can  determine retention of topology after radiation damage, recovery rates of global conformation, repair enzyme accessibility as well as impose limitations on detection of lesions.  Chromatin structure may also influence the distribution of radiation-induced  D N A lesions within cells. Detection of structural features of cell types that influence response to radiation would be of practical importance in designing predictive assays for tumour response to treatment.  In conjunction with the comet assay, a rapid method of D N A damage  detection in individual cells, the effects of radiation could be more accurately predicted by assessing not only the quantity of lesions produced but how individual cells 'cope' with X-ray insult, particularly double-strand breaks which cause lethal chromosomal aberrations. To examine the influence of variations in chromatin conformation on response to D N A double-strand breakage, a restriction endonuclease method was developed and optimized as an adjunct to comet analysis.  EcoRl cleaves double-stranded D N A at  defined sites within the genome. This enzyme was chosen to create DSBs for two cell lines which varied widely in radiosensitivity: Chinese hamster V79 lung fibroblasts and human B lymphoblastoid TK6 cells.  Chromatin organization was manipulated by  increasing NaCl concentration from 0 to 3 M to progressively remove different histone and non-histone proteins.  This approach provided a method to assess the possible  importance of accessibility of D N A recognition sites to the enzyme. For these cell lines,  Ill  no significant differences were observed, suggesting that accessibility to this restriction enzyme was not affected by differences in chromatin "packaging" that have been observed for these cells using other methods. However this general technique provides a potentially useful method for further restriction enzyme or D N A repair enzyme studies. As a related study, D N A repair enzymes for detection of base damage produced by ionizing radiation were examined for their ability to detect additional D N A damage in irradiated cells prepared as comets. Efforts were first made to optimize lysis and enzyme treatment conditions for V79 cells. For this project, alkali lysis and electrophoresis were necessary to detect single-strand breaks created when an endonuclease cleaves the phosphodiester backbone at sites of base damage. bacterium Micrococcal  A crude extract prepared from the  leuteus was examined for its ability to recognize ionizing  radiation-induced base damage and to produce additional SSBs in the comet assay. While additional damage was observed, there was also an increase in damage in unirradiated samples.  background  Since the crude extract may contain endogenous  nucleases that might mask the effects of base damage detection enzymes, a purified sample of Endonuclease III was then obtained and analyzed using the same method. However, in spite of considerable effort, no additional D N A damage was detected in irradiated cells exposed to Endo i n , suggesting that unknown factors may be preventing access of the enzyme to base damage sites or that conditions chosen for this study inadvertently affected enzyme activity.  iv  TABLE OF CONTENTS Page ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  vii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGMENTS  xii  DEDICATION  xiii  1.  INTRODUCTION  1.1  D N A Damage: Effects and Significance  1  1.2  Methods used to detect D N A damage in mammalian cells  2  1.3  The Comet Assay: Background  3  1.4  D N A Structure and Chromatin Conformation  4  1.4.1 Organization of D N A into higher order structures  4  1.4.2 Use of salt extraction to probe chromatin structure  8  1.4.3 Restriction enzymes as probes of chromatin structure The mechanism of action of EcoRl 1.4.4 Evidence for the influence of chromatin structure: radiation response 1.5  10 15 18  Detection of base damage using the comet assay  21  1.5.1 Radiation-induced damage to D N A bases  21  1.5.2 Methods for the detection of base damage  22  1.5.3 The base excision repair (BER) pathway  25  V  Page 2.  METHODS A N D MATERIALS  2.1  Cell Culture and Handling  27  2.2  Irradiation and Dosimetry  27  2.3  Comet Assay Technique  30  2.3.1 Preparation of slides  30  2.3.2 Alkaline lysis  31  2.3.3 Neutral lysis  32  2.3.4 Factors influencing migration during electrophoresis  32  2.3.5 Comet collection  32  2.3.6 Comet analysis  33  2.4  EcoRl Treatment of Cells  34  2.5  Detection of base damage using the comet assay  35  2.6  Statistical Analysis  38  3.  RESULTS  3.1  X-ray-induced killing and D N A strand breaks in V79 and TK6 cells using  3.3  the comet assay  40  Base damage detection using the comet assay  54  3.3.1 Background damage  54  3.3.2 Detection of damage by Micrococcal Endonuclease  57  vi  Page 4.  DISCUSSION  4.1  Influence of chromatin structure on radiation response  77  4.2  Detection of base damage using the comet assay  83  5.  S U M M A R Y A N D CONCLUSIONS  5.1  ZscoRl-induced damage using the comet assay  89  5.2  Base damage using the comet assay  90  6.  REFERENCES  92  A P P E N D I X 1 Fcrit and tcrit values for A N O V A and t test statistical analysis  115  Vll  LIST OF TABLES Page  Table 1.1:  Methods used to detect D N A DSBs in mammalian cells  3  Table 1.2:  Effect of salt concentration on D N A conformation  10  Table 2.1:  Dosimetry of 250kV X-ray Unit (0.5mm Cu filter)  28  Table 2.2:  Description of buffers for enzymes used to detect base damage  37  Table 3.1:  X-ray-induced SSBs detected by alkaline comet assay for V79 and TK6 cells  40  X-ray-induced DSBs detected by neutral comet assay for V79 and TK6 cells  41  Table 3.2:  Table 3.3:  Clonogenic survival data for V79 and TK6 cells damaged by X-rays  41  Table 3.4:  Effect of lysis solvent on accessibility to EcoRl in V79 cells  43  Table 3.5:  EcoRl dose response for V79 and TK6 cells following 15 Gy irradiation and 1 hour repair  45  Effect of salt concentration using 0.01 U / | i L EcoRl following 2Gy irradiation on V79 and TK6 cells  47  EcoRl dose response for cells permeabilized by 1% TX-100/ 2mM N a E D T A prior to enzyme treatment  50  Table 3.6:  Table 3.7  2  Table 3.8  EcoRl dose response for cells lysed in 1.2M NaCl/2mM N a E D T A prior to enzyme treatment 50  Table 3.9:  Comparison of correlation between mean tail moment decrease and D N A content loss for V79 and TK6 cells lysed under high salt conditions  52  Effect of salt extraction using 0.01 U/uL EcoR 1 for V79 and TK6 cells  54  Table 3.10:  2  viii  Page  Table 3.11:  Influence of lysis solution on untreated cells: background damage measurement  57  Qualitative comparison of results using different lysis solutions and application techniques  60  Table 3.13  Qualitative comparison of enzyme application techniques..  60  Table 3.14:  Base damage detected using l%TX-100/2mM N a E D T A / 1.2M NaCl lysis solution with variable concentrations of N a E D T A in reaction buffer  61  Base damage detected using 1 %TX-100/2mM N a E D T A / 1.2M NaCl lysis solution with 60mM N a E D T A in reaction buffer  64  Base damage detected using 1% TX-100/100mM N a E D T A / lOmM Tris/2.5M NaCl @ pH 10 lysis solution with variable concentrations of N a E D T A in reaction buffer  66  Effect of Concentration of N a E D T A in Reaction Buffer Using 1% TX-100/100mM Na EDTA/1 OmM Tris/2.5M NaCl @ pH 10 lysis solution  68  Base damage detected using l%TX-100/30mM N a E D T A / 1.2M NaCl lysis solution with I m M N a E D T A in reaction buffer  71  Base damage detected by Endonuclease III  71  Table 3.12:  2  2  Table 3.15:  2  2  Table 3.16:  2  2  Table 3.17:  2  2  Table 3.18:  2  2  Table 3.19:  /  Table 3.20:  Base damage detected by hot alkali treatment  74  Table 3.21:  Summary of Ness/Nssb for all base damage detection methods  76  Table 4.1:  Summary of studies of effect of conformation on D N A on radiation response Summary of studies of base damage detection using endonucleases  Table 4.2:  Table 4.3:  Methods used to detect SSBs in mammalian cells  83 87 88  ix  LIST OF FIGURES Page  Figure 1.1:  Schematic of higher order chromatin structure  7  Figure 1.2:  Effect of salt concentration on D N A conformation  11  Figure 1.3:  Mechanism of cleavage of restriction enzyme EcoRl  19  Figure 1.4:  Base excision repair pathway (BER)  26  Figure 2.1  Dosimetry of 250 k V X-ray unit (0.5mm Cu filter)  29  Figure 2.2:  Comet images and description of tail moment  34  Figure 2.3:  Schematic of EcoRl protocol  36  Figure 3.1:  Comparison of X-ray-induced SSB and DSB with clonogenic survival curves for V79 and T K 6 cell lines  42  Figure 3.2:  Effect of lysis solvent on accessibility to EcoRl in V79 cells  44  Figure 3.3:  EcoRl dose response for V79 and T K 6 cells following 15Gy irradiation and 1 hour repair Effect of salt concentration using 0.01U/|iL EcoRl following 2 Gy irradiation on V79 and TK6 cells  48  Bivariate plots of mean tail moment and D N A content comparing response to 2Gy irradiation for V79 and TK6 cells lysed under two different conditions  49  .EcoRl dose response curves for V79 and TK6 cells lysed in low and high salt conditions prior to enzyme application  51  Fraction of D N A content lost as a function of dose and correlation with mean tail moment decrease for V79 and T K 6 cells  53  Effect of salt extraction using 0.01U/|iL E c o R l for V79 and TK6 cells :  55  Figure 3.4:  Figure 3.5:  Figure 3.6:  Figure 3.7:  Figure 3.8:  46  X  Page  Figure 3.9:  Effect of salt extraction using X-rays on 4 cell lines  56  Figure 3.10:  Base damage detected as a function of M.luteus crude extract concentration with N a E D T A in reaction buffer using 1% TX-100/2mM Na EDTA/1.2M NaCl lysis solution  62  Effect of N a E D T A concentration in reaction buffer on 5pX of M.luteus crude extract using l%TX-100/2mM N a E D T A / 1.2M NaCl lysis solution  63  Base damage detected with 50|iL M. luteus crude extract in conjunction with 60mM N a E D T A in reaction buffer using 1% TX-100/ 2mM Na EDTA/1.2M NaCl lysis solution  65  Base damage detected as a function of M. luteus crude extract concentration with 10 m M N a E D T A in reaction buffer using 1% TX-100/100mM Na EDTA/1 OmM Tris/2.5M NaCl @pH 10 lysis solution  67  Effect of N a E D T A concentration in reaction buffer on 2|iL and 4|lL M. luteus crude extract using 1% TX-100/100mM N a E D T A / lOmM Tris/2.5M NaCl @ pH 10 lysis solution  69  Summary of effect of N a E D T A concentration in reaction buffer on M luteus crude extract concentration as a result of lysis, condition  70  Base damage detected as a function of Mluteus crude extract concentration using 1% TX-100/30mM Na EDTA/1.2M NaCl lysis solution  72  Figure 3.17:  Base damage detected using Endonuclease III  73  Figure 3.18:  Base damage detected using hot alkali treatment  75  2  2  Figure 3.11:  2  2  Figure 3.12:  2  2  Figure 3.13  2  2  Figure 3.14:  2  2  Figure 3.15:  Figure 3.16:  2  2  LIST OF ABBREVIATIONS  bp  Base pair  D  Daltons  DNase  Deoxyribonucleic nuclease  DSB  DSB  Endo HI  Endonuclease i n  Exo HI  Exonuclease IH  kb  kilobase  M . luteus  Micrococcal luteus  MEM  Minimal Essential Media  MNase  Micrococcal nuclease  MTM  Mean Tail Moment  NaCl  Sodium Chloride  NaOH  Sodium Hydroxide  Ness  Number of enzyme-sensitive sites  Nssb  Number of SSBs  PBS  Phosphate Buffered Saline  RE  Restriction Endonuclease  Sarc  N-lauroyl sarcosine (sodium salt)  SDS  Sodium dodecyl sulfate (sodium salt)  SSB  SSB  TX-100  Triton X-100 (non-ionizing detergent)  Xll  ACKNOWLEDGEMENTS I wish to thank W i l Cottingham for organizing my paperwork, of which there was a great deal. I appreciate Susan MacPhail, Dr. Judit Banath and Charlene Vikse for growing my cells and showing me general lab technique, and I am especially grateful to Dr. Peter Johnston for extremely helpful advice and assistance. I was also fortunate to have an incredibly understanding and patient supervisor, Dr. Peggy Olive, whose insight and humour I will always remember with fondness. The ongoing encouragement of Dr. Helen Freedhoff, Janet Johnson, my wonderfully sane roommate Selvyn Phinn, my confidante and masseuse, Kappa, meant a great deal, and of course, this degree would not have been possible to achieve without the love and support of my parents, Ruth and Nelson Thompson. Lastly I wish to thank Michele Mossman, who not only was with me every step of the way but also taught me how to live. Let the laughter continue....  xiii  This thesis is dedicated to the memory of my grandmother, Vera Mary Kirby-Tudhope  Introduction  1.  INTRODUCTION  1.1  DNA Damage by Ionizing Radiation: Effects and Significance  1  Within about one year of Roentgen's discovery of a new form of radiation in 1895, X rays were used in the treatment of cancer. Since that time, techniques have continued to evolve which harness the 'invisible light' for the purposes of diagnosis, therapy and discovery. Of principle importance from the standpoint of radiotherapy is the predictability of tumor response to ionizing radiation, which derives from assessment of damage induced by interactions of x-rays with biological targets. Detailed explanations of energy loss mechanisms are addressed by Hall [1994]. X-rays are a type of electromagnetic radiation with wavelengths ranging from lxlO" 3  lOnm. They can be thought of as a stream of photons and are capable of producing direct or indirect effects in biological targets, the latter through intermediate free radicals (reviewed by von Sonntag [1987]. D N A is at the top of the hierarchy of targets responsible for loss of reproductive cell death [Okada, 1970; Painter, 1980; Gentner and Paterson, 1984; Rauth, 1987; Hall, 1994] and so an understanding of damage contributing factors is essential for developing predictive assays and prescribing effective therapy.  Insult to D N A includes  backbone lesions in the form of both SSBs (500-1000/cell/Gy) or double strand breaks (2540/cell/Gy) in addition to base damage (1000/cell/Gy) and alkali-labile sites (200300/cell/Gy), so named for their appearance as strand lesions under alkaline conditions [Kubo et al, 1992; Wallace and Ide, 1990]. Double strand breaks (DSB), if left unrepaired, are considered to be the 'lethal lesion' often leading to chromosomal aberrations and loss of genetic information [Bryant, 1984a,1985; Natarajan and Obe, 1984; Blocher and Pohlit,1982; Frankenberg et ah, 1981]. However, misrepair of any of the above can further lead to  Introduction  2  mutations and fragmentation, resulting in immediate or eventual loss of reproductive integrity. The nature and extent of radiation induced D N A damage vary considerably in cells of different genetic constituency, tissue type, and cellular environment [Fertil and Malaise, 1985; Storer, 1982; Olive, 1994a, 1994c,]. Factors implicated in radiation response include oxygenation status, repair fidelity, cell-cell communication and intrinsic 'packaging' of chromatin, a kaleidoscope of influences which determine the outcome of cancer treatment. Clonogenic survival assays performed in conjunction with measurements of induced damage and strand break rejoining kinetics have proven useful in defining the range of radiosensitivities [reviewed in Olive et al., 1994c]. While it is held by some that initial numbers of double strand breaks and/or rejoining rates correlate with tumor response, [Radford, 1985; Kelland et al, 1988; Wlodek and Hittelman, 1987,1988a,1988b; Schwartz et al., 1996,1992], many others have concluded that there is no such relationship for some cell lines and suggest that it is the 'presentation' of damage that differs amongst cells[Iliakis, 1988; Olive,1992a; Olive et al., 1994c].  The controversy surrounding the issue of  radiosensitivity has ignited interest in chromatin conformation, its role in repair and influence on lesion detection.  The ability to discern structural differences should lead to a greater  understanding of factors important to repair and recovery of higher order conformation, and by extension may be useful in predicting therapeutic outcomes.  1.2  Methods Used to Detect DNA Damage in Mammalian Cells A variety of methods has been developed to measure D N A DSBs in mammalian cells  (Table 1.1). The sensitivity for detecting breaks is similar for all of the methods currently  Introduction  3  available, however, the effort and expense involved in performing the assays differ. Extensive reviews of techniques are given by Olive [1997], Nunez et al. [1996] and Elia et al. [ 1991].  Table 1.1: Methods Used to Detect DNA Double Strand Breaks in Mammalian Cells lixpense  Effort  Reference  125-2500  Approx Time Required 24 hours  Moderate  High  Kohn,1991  125-5000  96 hours  Moderate  High  Blocher, 1982  125-2500  24-96 hr.  Low  Low  125-7500  24 hours  Moderate  Mod.  Blocher,1982; Stamato and Denko, 1990 Olive et al. 1991  Method  Scnsiti\ ily (breaks/cell)  Neutral filter elution Neutral sucrose gradient sedimentation Neutral gel electrophoresis Neutral Comet Assay  "Estimates of range of sensitivity are based on response to ionizing radiation, with 1 Gy producing 25 DSB/diploid cell Effort reflects technical proficiency required and amount of time required per sample. From Olive, 1997.  1.3  The Comet Assay: Background The comet assay was first introduced as a microelectrophoretic technique for the  direct visualization of D N A damage at the level of a single cell [Osling and Johanson, 1984]. Since that time, this versatile assay has been optimized with respect to lysis solutions and times, electrophoresis conditions, effects of salt concentration and nature of intercalating dye [Olive et al, 1990b, 1994b; Singh et al, 1994, 1988; reviewed by Fairbairn et al, 1995 and Klaude et al, 1994]. The development of image analysis methods allowed an objective measure of D N A damage [Olive et al, 1990b]. To perform this method, single cells are  Introduction  4  embedded in agarose, lysed and subjected to an electric field. This technique exploits the negatively charged property of the phosphate groups on the D N A molecule and pulls D N A strands toward the anode in a size-dependent fashion, based on the maneuverability through the agarose pores. The resulting pattern, visualized using fluorescence dyes that bind to D N A , closely resembles a comet; the shorter pieces accumulating in the 'tail' region (Fig 2.2). Software written by Ralph Durand of the B C C R C translates intensity and relative location of light excitation into assessment of D N A strand breaks in terms of features that include D N A content, tail moment, and percentage of D N A in the tail, defined in Chapter 2. A great deal can be inferred from the comet data in addition to strand breaks, such as hypoxic status of tumors, growth fraction of a population, presence of apoptotic cells, and effect of chromatin organization [Olive et al., 1992a]. A n important advantage of this method over other strand break assays is that relatively few cells (less than 10,000) are required and can be obtained by fine needle aspiration or from a single drop of blood. Analysis of comets can be performed rapidly (up to 800/hr) and the method does not require radiolabeling so that non-cycling cells can be analyzed. The sensitivity of the assay is about 50 SSB/cell/Gy and 125 DSB/cell/Gy, comparing favorably with other methods, especially for low dose detection (Tables 1.1 and 4.3).  1.4.  DNA Structure and Chromatin Conformation  1.4.1  Organization of DNA into Higher Order Structures Deoxyribonucleic acid (DNA) exists as two phosphodiester backbones containing  repeating nucleosides held together largely by complementary base paired hydrogen bonds [for details see Neidle, 1994; van Holde, 1988]. The antiparallel strands of free p-DNA in  Introduction  5  solution are twisted to form a double helix with periodicity of 3.6nm (10.5bp/turn) and helical rise of 0.34nm(lbp separation).  The spatial arrangement created by stacking of  hydrophobic purines and pyrimidines in nearly planar orientation perpendicular to the long axis generates both a minor and major groove. Sequence dependent structural variations (such as poly A) have been found to facilitate bending, which is necessary for its compaction and in addition a multitude of morphological configurations are allowed by six possible rotational angles of the backbone, several base presentations such as propeller, helical twist and roll as well as sugar puckering. At this basic level of chromatin organization alone, there is an incredible capacity for dynamic and fluid rearrangement, contributing further to the diversity of type and strength of interaction with protein. The nature of higher level D N A packaging as seen under physiological conditions is of key interest in designing predictive assays, so a better understanding of chromatin organization is needed. Consistent among cell lines is the property that 146 bp of D N A are wrapped 1.8 times around a core histone octomer which is a trimeric unit of a tetramer of 2 H3-H4 histones flanked on either side by 2 dimers of H2A-H2B. (For details about nucleosomal structure and its interaction with D N A refer to Lowary and Widom[1997]; Pruss et al [1996, 1995]; Arents and Moudrianikis [1993]; reviewed by Moudrianakis and Arents [1993]; Hayes[1990]).  The properties of individual histones are well characterized with  respect to their amino acid composition, electrophoretic mobility and other attributes [Lubomir et al.., 1989] but of essential importance to this study is their interaction with D N A in terms of charge, affinity and positioning. The positively charged histone octomer at physiological cation strength is neutralized by contact with the negatively charged phosphate groups of D N A , however the association is far from rigid; it has been found that 5-20% of octomers dissociate spontaneously at physiological salt concentrations [Stein, 1979; Stacks  Introduction  6  and Schumaker, 1979; Eisenberg and Felsenfeld, 1981; Yager and van Holde, 1984]. The forces which 'fold' the D N A into chromatin fibers include long and short range nucleosomal and non-histone protein interactions of ionic, hydrogen, van der Waals and hydrophobic nature in addition to the obvious contribution of histone H I , which associates both with linker D N A and the nucleosome core [Widom, 1989; Zlatanova, 1996,1991; Hayes, 1991]. Histone H I is thought not only to stabilize tight packaging but also to serve in an immobilization capacity, playing a role in the gene regulation of transcription and repair. The total 10,000-fold compaction of D N A in a chromosome is made possible by successive folding of nucleosomal D N A into 30 nm fibers which are attached to the nuclear scaffold at matrix attachment regions via D N A sequences [Stein and Berezney, 1996; Izaurralde et al, 1988; Cook and Brazell, 1975, 1988]. As reported by many [Berezney et al., 1995,1991; Mirkovitch et al., 1984, 1987, 1988; Pienta and Coffey, 1984; reviewed by Davie, 1995], the nuclear scaffold constrains chromatin in a number of loop domains at these sites and clusters containing 50-60 loops are thought to be involved in sensitivity to ionizing radiation for some lines [van Rensburg et al., 1987]. Nuclear matrix-associated proteins and changes in D N A supercoiling have further been shown to correlate with radiosensitivity [Malyapa et al., 1994, 1996a, 1996b; Taylor et al, 1991; Vaughan et al, 1991; reviewed by Roti Roti et al, 1993]. Chromatids are formed in a solenoid or ribbon fashion with approximately 30 rosettes incorporated into each coil. Recently, ionizing radiation has been used to probe chromatin organization by measuring D N A fragment sizes after irradiation, and matching fragment size with different models for chromatin organization [Holley and Chatterjee, 1996; Rydberg and Chatterjee, 1996]. A schematic of the higher chromatin organization is shown in Fig. 1.1.  Introduction  Higher Order Chromatin Structures (periodicities in parentheses) DNA Double Helix  Chromosome (approx. 250 Mbp)  Figure 1.1: Schematic of Higher Order Chromatin Structure.  Introduction  8  The spacing of nucleosomes is thought to ultimately affect higher order packaging by influencing the binding of non-histone proteins [Blank and Becker, 1996; Hayes, 1990; Thoma and Simpson, 1988; Thoma and Roller, 1981]. In addition, a highly variable degree of 'quantization', which refers to saturation and repetitive patterning, is reported among lines, determining the structural regularity of the condensed 30nm fiber [Yang et al., 1994; Yao et al., 1993; Woodcock et al, 1993; Widom, 1992;  reviewed by Simpson, 1991].  The  nucleosome repeat length (NRL) consists of the 146bp D N A plus variable linker length; NRLs of up to 268 bp have been reported [van Holde, 1988]. A second element which has deserved attention is octomer positioning, and the debate continues as to whether electrostatic or sequencing effects dominate [Blank and Becker, 1995, Kralovics et al. 1995; Prunell and Kornberg, 1978]. However certainly deformational anisotropy (bendability) plays a vital role in octomer-DNA interaction as well as accessibility to repair enzymes [Kralovics et al, 1995; Yao et al, 1991; Calladine and Drew, 1986; Drew and Travers, 1985].  1.4.2  Use of Salt Extraction to Probe Chromatin Structure One of the many techniques used to render D N A accessible to damage or to analysis  of that damage is salt extraction; for a review of other methods see Fletcher [1996]. This technique has proven to be compatible with analysis of D N A damage using the comet assay, and can allow progressive lysis in stages by increasing salt concentration [Olive and Banath, 1995]. Chromatin undergoes reversible alteration in response to increasing ionic strength [Yager and van Holde, 1984], however it is possible that the organization of D N A in the nucleosomal core differs substantially from that at low ionic strength [Drew, 1991]. At low  Introduction  9  cation concentrations (ie at 1 mM), the chromatin is in a highly extended form, nucleosomes attach like 'beads' on a string to D N A which is anchored to proteins of the nuclear matrix. The association of histone H I , apparently is required for further condensation into 30nM fibers [Widom, 1989; Zlatanova, 1991; Freeman and Garrard, 1992], although studies involving higher cation concentrations have shown that compaction, although more weakly bound, can be achieved in its absence [Lewis et al., 1975; Renz et al. 1978,1977] and this is thought to be related to D N A properties [Thoma and Roller, 1977; Thoma 1992, 1986]. As physiological conditions are met (150-200mM NaCl), the 30nM fiber further folds into solenoidal arrangements, which are densely packed. At this level of organization, there is minimal accessibility to damaging agents. Figuyre 1.2 illustrates D N A conformation at this level of salt concentration. Further increases in salt level disrupt the charge neutralization that the histone and non-histone proteins effected; the D N A apparently favors interaction with salt and proteins successively dissociate. information. NaCl levels of  Table 1.2 summarizes the following  0.35, 0.6, 1.2M and 2.0M extract respectively non-histone  proteins, H I , the H2A-H2B dimer and finally the H3-H4 tetramer. At 0.6M, nucleosomal 'slide' has been reported [Lohr et al.., 1977, 1979] and at 0.8M, the complete histone octomer can exchange to exogenous D N A [Germond et al.., 1976]. Removal of the nuclear matrix requires either a very high salt presence (>3M) or a combination of high temperature and a strong ionic detergent such as SDS. The removal of histones resulting in compensatory supercoiling of the D N A is a factor influencing outcome of damage caused by radiation and enzymes.  Further studies on chromatin organization at high salt concentrations and  temperatures have been done by Bashkin et al., 1993.  Introduction 10 The importance of this effect is well documented by those interested in the cellular outcomes for damaging agents. Olive et al [1995] found that the ability of ionizing radiation to produce DSB increases approximately 20 fold upon depletion of histones. Evidently, as stated by van Holde [1988], these structures play a vital role in protection against strand breaks in addition to potentiating repair. This is confirmed by findings using O H radicals [Nygren et al., 1995; reviewed by Ward, 1990] to probe chromatin structure.  Table 1.2: Effect of Salt Concentration on Conformation of DNA INaCIl  Protein Extracted  0.0 m M 1 mM  Topological Effect Chromatin highly compacted Chromatin expanded; nucleosomes and H I unaffected  0.35 M 0.6 M  many non-histone proteins HI and other non-histone proteins  1.2 M 2.0 M  H2A and H2B dimers H3A and H3B dimers  Chromatin extended like 'beads on a string' D N A attached to nuclear scaffold in loop domains at 30-100kb regions  The effect of salt on restriction endonucleases is similar, in terms of disruption of electrostatic balance. Salt stimulates binding up to 150mM and inhibits it beyond. It is thought that the salt sets up effective 'shields' of the negative charges on the enzyme complex, ensuring maximal binding with D N A to the basic elements [Rosenberg, 1980].  1.4.3  Restriction Enzymes as Probes of Chromatin Structure Res, particularly type II which recognize specific base pair sequences, are valuable  research tools in molecular biology. Type II cutters include those which produce 'blunt' or 'staggered' representations (Fig. 1.3) and the latter type such as produced by EcoRl, result in cohesive or 'sticky' ends, useful in construction of chimeric D N A ideal for recombinant D N A research. In addition to use in sequencing, mapping, genome characterization,  Introduction 11  EXTENDED  CORE HISTONES  HI HISTONES LOW IONIC STRENGTH  PHYSIOLOGICAL IONIC STRENGTH  NUCLEAR MATRIX  Figure 1.2: Effect of Salt Concentration on DNA Conformation. At cation concentrations below physiological levels, chromatin is in fully extended form. The addition of H1 helps compact nucleosomal DNA into 30nm fibers; chromatin is fully compacted at concentrations slightly above physiological ionic strength.  Introduction 12 identification and cloning, restriction enzymes can mimic the effects of radiation-induced damage in the form of DSBs. Res cause strand breaks only; isolation of this damage from peripheral base damage, repair, cross linking and apoptosis phenomena allows ideal probing of D N A conformation. In addition, as type II enzymes require no A T P for cleavage, they retain efficiency in a manipulated cell-free environment such is produced in conjunction with the comet assay. R E are purified from bacteria, in which their native purpose is to degrade foreign D N A within the cell based on a specific recognition sequence. The cell's own sequence is protected by methylation of bases, which has been shown in vitro to inhibit the activity of the restriction enzyme [Rosenberg, 1980]. Endonucleases are so named for their recognition and cleavage within (endo) a length of D N A , the key feature which allows information to be obtained on how the D N A is packaged along the entire length of the chain. Investigations of chromatin structure using enzymes have paved the way to a better understanding of the complex organization of D N A . Digestion and footprinting studies done using DNase I and Micrococcal nuclease (MNase) have revealed specific structural features such as nucleosomal positioning, dissociation and mobility, all of which are essential parameters of DNA-dependent reactions inclusive of regulation, transcription and repair. [Tanaka, S. Et al, 1996; Leuba et al, 1994; Jackson and Felsenfeld, 1985; Jalouzot, R., et al, 1980; Sollner-Webb et al, 1978]. This data is consistent with that obtained.using sitedirected hydroxyl radicals to map nucleosome position [Flaus et al, 1996]. The accessibility of these enzymes to cleavage sites, as seen by patterning on polyacrylamide gels, can infer the properties not only of nucleosomes, but also of higher order interactions such as H I and H5 positioning and the influence of histone tail domains [Staynov, D.Z.,1988; Lutter, 1979; Noll  Introduction 13 and Kornberg, 1977]. DNase I, interacting with the minor groove, introduces single strand breaks every lObp in linker D N A as well as D N A wrapped around the core wherever the minor groove faces outward. Thus this enzyme is a rotational specifier, dependent on the orientation of D N A .  MNase I, by comparison, yields translational information, cleaving  preferentially linker D N A , however it can also characterize the nucleosomal surface [Whitlock, 1977]. Aside from differences seen in D N A lengths, temporal variations have been observed amongst different lines, mainly due to occlusion of enzymes by histones, adjacent D N A turns, deformation and sequence specifications. In addition, Jalouzot at al [1980] and others have found enzymes useful in determining structural changes resulting from cell cycle position. Not surprisingly, the more highly condensed metaphase cells have been found to resist degradation, unlike the more open S-phase configurations.  These  findings are in excellent agreement with radiation effect data related to cell cycle. Interest in double strand lesions and their repair has led to studies using enzymes of a restriction nature, which cleave at certain sequences in a double stranded (for some) fashion. Employing such a damaging agent, despite its suspected inability to cleave nucleosomebound D N A , provides insight into accessibility of D N A to repair enzymes, largely because repair processes involve rearrangement or dissociation of the octomer.  In addition, key  properties of nucleosomes (ie: phasing) are known to differ widely amongst cell types [Fletcher, 1996; Cavallii and Thoma, 1993; Siderik and Smerden, 1987]. Restriction enzyme studies involving chromatin structure via band patterning have proven to be informative about both chromatin organization in cell types and cleavage patterning demonstrated by individual endonuclease [Bianchi and Bianchi, 1987].  Introduction 14 Restriction enzymes are ideal for mimicking  damage resulting from ionizing  radiation, in that they can produce either blunt or staggered cohesive ends, interstrand and protein-DNA crosslinks and can exhibit clusters of damage not unlike those associated with secondary ionization events, resulting from the reaction mechanism described later in this chapter.  They do not, however, produce base damage or 'dirty' ends (breaks in sugar  residues) , as the breaks result from hydrolysis of the phosphodiester backbone and it is thought that the mechanism of repair deviates from the pathway used to repair radiationinduced lesions [Bryant, 1988a].  REs have also been useful in apoptosis studies; the  resulting damage closely resembles degradation patterns formed during the collapse of chromatin into fragments. The first use of restriction endonucleases in cytogenic work was in barley root tips treated with Hind II and Hind IH. Since that time, extensive studies have revealed increased chromosomal aberrations arising from double strand breaks after enzymatic treatment, and repair process explanations are being actively sought [Bryant, 1994; reviewed by Bryant, 1988a, 1988b and by Bryant and Johnston, 1993a.; Powell, S.N. et al., 1994; Cortes and Ortiz, 1992, 1991; Costa and Bryant,1991a, 1991b, 1988; Natarajan et al., 1985; Natarajan and Obe, G.,1984]. This work ultimately seeks to explain ranges in radiosensitivity such as seen in DSB repair deficient ataxia telangiectasia (AT) cells.  Bryant has had some success  correlating such breaks in radiosensitive xrs5 vs parental resistant C H O K l cells using neutral filter  elution, however this finding  is not consistent  with  differences in  radiosensitivity among largely repair proficient cell types (see Olive, 1994c). R E studies to probe chromatin structure using band patterning have demonstrated not only differences in patterning among cell lines, but also of endonucleases [Bianchi and Bianchi, 1987].  Introduction 15 Interestingly, many of the groups active in this field have found that blunt-end producing endonucleases cause far greater numbers of aberrations than the more frequently occurring (by ionizing radiation) cohesive-end cutters. This is thought to be due to the more deletional nature of the break, that is, with cohesive end production, only one of the strands is missing information. Given the wealth of information on the subject of enzyme kinetics, specificity, damage potential and substrate affinity, it has been possible to both design a predictive strand break assay as well as understand the structural implications from analysis of the data. Essentially, the method developed in this study is an extension of work done using REs on cells with relatively intact repair capacity; the key difference is that static, not dynamic conditions are involved, in order to 'freeze' a population of cells possessing random structural arrangements.  Mechanism of Action of EcoRl EcoRl, purified from the bacteria Escherichia Coli is a type II restriction enzyme  comprised of a dimer of identical polypeptides each of M r 3 I K [Halford, 1983]. This enzyme recognizes a centrosymmetric palindromic hexameric sequence as detailed in Fig 1.2. It is thought that palindromic sequences have the potential to from cruciforms, which bend the D N A and contribute to inaccessibility in linker D N A . The mechanism of cleavage has been well investigated [Halford, 1983; Rosenberg et al., 1981, Rubin and Modrich, 1978, Modrich and Zabel, 1976] and it is known to be a staggered, usually two step single strand scission following hydrolysis of phosphodiester bonds, resulting in a double strand break at a defined distance from the recognition site. The two protein subunits interact symmetrically  Introduction 16 with the recognition site on D N A and each subunit requires a protein conformational change before cleavage is possible; this is referred to as the 'Kochland' mechanism of independent conformation.  D N A sequence recognition is mediated by 12 hydrogen bonds formed  between the purines in the recognition site and six amino acid residues in the dimeric endonuclease (one Glu and 2 Arg residues in each unit)[Frederick et al., 1984]. Factors influencing activity aside from temperature,  ionic strength  and pH  requirements are structural. Important parameters have demonstrated [Kessler, 1987] to be: 1. G C content and base distribution in natural D N A 2. 3. 4. 5.  Size of the D N A and cleavage frequency Length and base composition of flanking sequences Position of cleavage sites with respect to each other D N A tertiary structure  6. Modification of D N A (ie: methylation) and protein attachment The mechanism of action is a four step reaction (deduced by Modrich and Zabel, 1976; Rubin and Modrich 1978; Hinsch and Kula, 1981). Schematically it proceeds as: kl  E + S <=$> E-S k-1  k2  =>  k3  E-I => k5 •litl k-5 E +I  k4  E-P =>  E +P  The initial binding of the enzyme E to specific recognition sites of D N A substrate S, forming the Michaelis complex ES, is followed by cleavage of a first phosphodiester bond, resulting in open, nicked intermediate I, which can dissociate from the enzyme, resulting in a single strand lesion and free enzyme (step 5). Cleavage of a second phosphodiester bond results in completely digested D N A product P and its dissociation from the enzyme is the rate limiting step. There are two hypotheses of cleavage mechanisms which are supported by reaction profiles. The first posits that the enzyme dimer remains associated throughout both scission processes and the second allows for the dissociation step (k5); there is evidence that  Introduction 17 both processes occur. This contributes vastly to the heterogeneity within and amongst cells, especially for short incubation times. The enzyme obeys Michaelis-Menton kinetics of first order velocity with respect to endonuclease concentration. The overall reaction serves to produce predominantly double strand breaks and the enzyme, upon dissociation is free to make repeat cuts.  This is  important from the standpoint of assigning assay parameters such as incubation duration and concentration. The velocity of the reaction is linearly dependent on enzyme concentration in higher amounts, which is of importance in setting an acceptable range. The topological state of D N A is of key importance to cleavage by EcoRl.  The  primary determinant of the recognition specificity is the direct interaction with the complementary protein-filled major groove, which contains the recognition sequence, however, flanking backbone segments appear to modulate binding. Obviously, these flanking regions will differ in morphology amongst cell types based on sequencing effects and so this phenomenon could ultimately affect recognition. An understanding of how morphological variations in structure influence binding, and therefore potential cleavage, is crucial.  Crystallographic analysis, visualized by X-ray  diffraction methods of the synthetic oligonucleotide T C G C G A A T T C G C G (EcoRl cleavage site underlined), has revealed that a specific kinked D N A configuration, resulting from partial unwinding of the helix, is a prerequisite for formation of the enzyme-DNA complex. [Frederick et al., 1984]. This analysis, derived from extensive modeling of the dodecamer of Dickerson and Drew [1981] and later studies such as done by Patel et al. [1982], shows that systematic shifts in the coordinates of phosphate, sugar and base moieties are necessary for accessibility. In addition, the central block of the oligonucleotide is a 10.3bp per helical  Introduction 18 repeat, which is within the values of 10 to 10.6 bp per turn reported for D N A in nucleosomes [Lutter,1979]. A number of structural properties can in inferred from EcoRVs ability to bind, based on the Dickerson-Drew model. Characteristics of the model are that the narrow minor groove in the 5 ' A A T T region is of 3.2 A width (compared to 6.0A in naked |3 form DNA), a spine of hydration, (which no doubt influences 'packaging' in terms of association with the hydrophobic amino acids of proteins), specific base pair parameters, conformational angles and an observed 19° bending. Rather than elaborate on such features, it is sufficient to conclude that a great deal of reorganization of chromatin structure must be performed in order for EcoRl sites to be bound. It is reasonable to assume, from this theory, that results assessed in dynamic systems (such as those used by Bryant and his colleagues) will differ enormously from those evaluated under static conditions.  Such a theory is based on the  above models and it is likely that the most accessible regions of chromatin, either determined by sequence or by steric hindrance, are those which are flexible to dynamic interaction. This view is supported by the work of Saavedra [1990], where he found that the accessibility of D N A in vivo to polymerases was dependent on the degree of conformational mobility. Recently a model for gene regulation, designed by Polach and Widom [1995], presents evidence for regulatory protein accessibility being dependent on the dynamic and transient nature of nucleosomes.  1.4.4  Evidence for the Influence of Chromatin Structure: Radiation Response The evidence for chromatin structural influence on distribution and repair of ionizing  damage has been reported using a wide range of techniques [Nygren et al., 1995; Olive, 1995,  Introduction 19  A-A-T-T-Ci  "ft  5»i  iC-T-T-A-AtG«  3'i  I  Cohesive end /  ,.T!  iG I  III  i  +  •C-T-T-A-A  *  ' \  A-A-T-T-C I I I I | G  Cohesive end  Rqure 1 3: Mechanism of Cleavage of Restriction Endonuclease EcoRl. The endonuclease is shown to cleave in a staggered fashion at the sites within the 6 base-pair recognition sequence. Ranking sequences (not shown) are palindromic.  Introduction 20 1992a; 1991; Johnston and Bryant, 1994; Elia and Bradley, 1992; Ljungman et al, 1992, 1991a, Warters, 1992, 1990; 1991b; Yasui et al, 1987; Chiu et al. 1986, 1982a, 1982b, Wheeler, 1983; Mee, 1981]. Findings linking the non-random distribution of damage to chromatin conformation include radiosensitive increase in transcriptionally active D N A [Chiu et al. 1982] and histone depleted structures [Olive, 1995; Xue et al, 1994; Elia and Bradley, 1992; Warters and Lyons, 1992, 1990; Ljungman, 1991b]. The controversy that surrounds correlation of initial damage with radiosensitivity is largely based on reported differences seen using dissimilar detection methods.  The unresolved dilemma is whether  assays such as filter elution and nucleoid methods measure real differences (ie: number of lesions) or simply detect differences in the way chromatin responds in these methods. It is known, for example, that cells in S-phase contain replicating structures that affect the way the double strand breaks are interpreted by elution, sedimentation and gel electrophoresis [Sweigert et al, 1988; Okayasu et al, 1988; Olive et al, 1992d, 1991]. These associations with protein which inhibit migration may be related to radiosensitivity. Cook [1988] argues that chromosome morphology might be maintained in the presence of strong detergents, even after removal of histone and non-histone proteins, creating difficulties in assessing breaks. In both cases, results strongly implicate chromatin-dependent differences in radiosensitivity. Neutral filter elution results, such as those presented by Radford [1985], Kelland et al. [1988], Schwartz et al. [1991], and Wlodek and Hittelman [1987], indicate that D N A from radiosensitive cells elutes more rapidly from filters than D N A from resistant cells; this is thought to be due to greater availability of D N A for shearing following the increased expansion after lysis [Wlodek and Olive, 1990]. Resistant cells could contain some structure which is resistant to the lysis conditions typical of filter elution, but which is not stable under  Introduction 21 conditions of other strand break assays. Studies done using gel electrophoresis and neutral filter elution show an increased frequency of D N A strand breaks after exposure to p H 9.6 [Flick at al, 1989] and D N A strand break detection has been shown to be affected by eluting solution composition by Koval [1988b]. The neutral halo assay further seeks to explain 'packaging' based on changes in D N A supercoiling brought about by strand breaks. It has been proposed [Roti Roti et al., 1993; Roti Roti and Wright, 1987] that the nature and number of nuclear matrix attachment sites influences the extent of damage by rendering neighbouring loop domains more accessible and changing the rate of expansion of relaxed coils [Taylor et al., 1991; Cook and Brazell, 1975]. Scavenging studies have shown that higher order folding determines the amount of water available for free radical formation, leading to increased amounts of damage in certain types of cells [Oleinick, 1994].  In addition, through enzymatic probing, specifically using  restriction enzymes to mimic radiation damage, the way in which radiation sensitive and thereby accessible sites are distributed can provide insight into spatial and functional arrangement of chromatin.  1.5  Detection of Base Damage Using the Comet Assay  1.5.1  Radiation-Induced Damage to DNA Bases Oxidative base damage is part of the spectrum of damage caused by ionizing radiation  and is potentially lethal, owing to dramatic changes in the genetic code which arise from mutations, deletions and rearrangements of D N A ; they are also strong blocks to D N A synthesis in vitro [Schaaper et al., 1983 ].  Introduction 22 Damage to bases caused by oxygen free radicals include fragmented or ring-opened forms of pyrimidines and purines and oxidized aromatic derivatives with potential mispairing properties; in addition, bases may participate in DNA-protein crosslinks.  The most  frequently studied lesion has been the abasic (AP) site, since not only is it produced by ionizing radiation [von Sontag, 1987], chemical and alkylating agents [Loeb and Preston, 1986], but also is formed spontaneously under physiological conditions at a rate of 10,000/cell/day [Wallace and Ide, 1990; Kubo et ah, 1992]. Apurinic/apyrimidinic or abasic sugars (AP) sites are the result of hydrolysis of the N-glycosylic bond between the deoxyribose and the attached base.  1.5.2  Methods for the Detection of Base Damage Several methods are available to quantitate A P sites in D N A , either directly using  H P L C , or indirectly via single strand break analysis of lesions produced as a result of alkali treatment of A P site-containing D N A . However, results vary widely both amongst different techniques and even within those using the same methods due to mainly lysis conditions and detection sensitivity limits. There is speculation about differences in the intrinsic success between assays that require radiolabeling and those that do not; it is admitted, however, that labeling assays can 32  be cumbersome to set up [Bryant, 1988 ]. Weinfeld et a/.[1991]using a  P postlabehng  assay were able to quantitate A P sites at the femtomole level as were Talpaert-Borle and Liuzzi, [1983] using  1 4  C labeled methoxamine, however, large quantities of D N A were  required to achieve sensitivity. Kubo et al. [1992] had some success using a biotin-tagged reagent (ARP), which is specific for the aldehyde group, to quantitate A P sites using an Elisa-  Introduction 23 like microliter plate assay, however, due to biochemical interference blocking aldehyde accessibility, many forms of damage, such as thymine glycols and pyrimidine dimers could not be properly assessed. However, most base damage must first be detected using a damage specific endonuclease. A n alternative method of assessing base damage indirectly is performed using endonucleases, in which single strand breaks are created at sites of base damage.  Many  groups use crude extracts of M. luteus to produce single stranded lesions which are later assessed by sedimentation [Skov, 1984, 1979; Wilkins,1973; Schon-Bopp et al., 1977], alkaline elution [van Loon et al., 1993, 1991] or alkaline unwinding techniques [Bryant et al., 1978; Fohe and Dikomey, 1994]. A number of specific, separable gamma endonuclease activities have been identified [Schon-Bopp, 1977], and a Mg++ -independent gamma endonuclease of M.luteus has been purified approximately 1000-fold [Jorgensen et al., 1987]. M. Luteus, in crude fraction II form, has been found to cleave not only modified pyrimidines, but thymine glycols and urea residues in addition to apurinic sites [Jorgensen et al., 1988]. Synthetic indicators, which are purified to homogeneity, include Endonuclease HI (often in conjunction with formamidopyrimidine glycosylase (FPG) and exonuclease HI. Endo HI recognizes 5,6 dihydopyrimidine derivatives and A P sites; F P G proteins cleave at 7,8 dihydro-8-oxoguanine, formamidopyrimidines and A P sites; exo HI cuts at A P sites only (for extensive review of structure and function of endonuclease HI refer to Kuo et al., 1992; Kow et al., 1987; for exonuclease III see Mol et al, 1995; Kuo et al, 19 ). Presumably, almost all base damage can be detected using a cocktail of the above enzymes. Much evidence is cited of the ability to detect base damage via direct (HPLC and gas chromatography), or indirect alkali means and accumulated damage by the use of repair  Introduction 24 inhibitors  like  l-(3-D-arabinofuranosylcytosine (Ara C), hydroxyurea  and  9-|3-D-  arabinofuranosyladenine (Ara A) [ Iliakis et al, 1989]. However, these methods can only detect differences in total number of breaks within a population. A notable exception is the comet assay, which allows examination of response at a single cell level. Recently, base damage detection has been described by Collins et al. [1995, 1993] using this method. Alkaline filter elution, sucrose gradient sedimentation and unwinding techniques, by comparison, provide only a measure of damage to the entire population. Alkaline sucrose sedimentation, which has received the most attention for base damage detection of this kind, involves degradation, removal of D N A bound proteins, separation of strands and release of D N A onto a sucrose gradient; the size distribution of fragments determines the number of breaks.  Drawbacks include damaging or variational effects of rotor speed, wall effects,  gradient capacity, gel formation and rate of D N A unwinding; in addition the study of high molecular weight D N A is often a problem (see Lett [1981] and Leroy et al. [1996] for reviews). The alkaline elution technique appears to have greater sensitivity [Koch, 1994], however, long lysis times in alkali are thought to create higher background breaks due to alkali-labile sites.  The unwinding technique, which uses very short duration of alkali  exposure, fares better in this respect and is also more sensitive in terms of low end detection of breaks. The problem with this assay is that it exploits the rate of denaturation, and results are known to fluctuate greatly [ Anhstrom, 1988].  This technique determines relative  amounts of DSB/SSB by hydroxyapatite chromatography following lysis and neutralization. It has been performed with varied success by Fohe and Dikomey [1994] and also Bryant et al.  Introduction 25 [1978] using different methods of enzymatic application, a factor crucial in determining heterogeneity of response, as seen using the comet assay. A l l of the above alkali methods show a 10-fold decrease in base damage from H P L C results in addition to being influenced by alkali-related effects [Collins, 1996]. Interestingly, it has been reported by Fuciarelli et al. [1990] that yields of base products detected using gas chromatography differ according to gassing conditions. They believe that amount of base damage is affected by both the conformation of D N A as well as oxygenation status. Oxygenation level influence on base damage has also been reported by Skov [1979, 1984] and others. A summary of techniques used to measure SSBs is presented in Table 4.3.  1.5.3  BER pathway Base damage is repaired by both nucleotide excision repair and base excision repair  (BER) pathways [for reviews see Seeberg et al, 1995; Sancar, 1995; Barnes, 1993; Ward, 1991; Teebor, 1990 (HPLC); Lindahl, 1990,1982, 1979; Sancar and Sancar, 1988]. Essentially, the modified base is removed by hydrolysis of the N-glycosylic bond between the deoxyribose and base by D N A glycosylase. This reaction produces an A P site, which is cleaved by phosphodiester hydrolysis on the 5' (AP lyase) or 3' (AP endonuclease) side of the abasic deoxyribose (Fig. 1.4). This initiates the subsequent replacement of the deoxyribose with a nucleotide by polymerases and ligases. Endo III, like the gamma endonuclease purified from Micrococcus Luteus, appears to act as both a N-glycosylase and an A P endonuclease/lyase, recognizing a wide range of oxidized base damage as previously outlined. Both enzymes show no preference for supercoiled vs relaxed D N A , however they are highly dependent on pH and temperature conditions.  Materials and Methods 26  Figure 1.4: The base excision repair (BER) pathway. The nucleotide structure drawn is assumed to be part of a longer double-stranded DNA. Repair by DNA glycosylases without associated AP-lyase activity generally follows the pathway using AP-endonuclease and 5'-deoxyribophosphodiesterase activity. However, repair with N-glycosylases having associated AP-lyase activity uses the 3'-phosphodiesterase activity of the AP-endonuclease to make a single nucleotide gap that is filled by DNA polymerase and ligase.  Materials and Methods 27  2. MATERIALS AND METHODS 2.1  Cell Culture and Handling Chinese Hamster V79 lung fibroblasts were maintained as exponentially growing  monolayers in minimal essential medium (GIBCO) supplemented with 10% fetal bovine serum (Sigma Chemical Co., Mississauga, Ont.). Cells were grown up in 100x20mm tissue dishes (Falcon#3002) at 37° and 5% C O 2 .  Prior to experimentation, cells were typsinized  (0.1%) for 5 minutes, resuspended in fresh medium and incubated at 37° for an additional 10 minutes to allow repair of possible trypsin-induced damage.  T K 6 human lymphoblastoid  cells were maintained in suspension in RPM1 medium (GIBCO) containing 10% FBS in vented tissue culture flasks.  Both lines were subcultured three times per week and  consistently 24 hours prior to experimentation to ensure asynchronous exponentially growing populations. Plating efficiency of both cell lines was routinely > 0.7. V79 cells have a tendency to self aggregate, making them ideal for spheroid growth, but difficult for single cell studies and so were subsequently pipetted several times prior to counting.  As a check, the Coulter counter provides information via signal threshold  regarding the size and number of particles, ensuring that a uniform distribution of single cells was prepared. Typically, two measurements of cell concentration were taken, averaged and multiplied by a dilution factor.  Suspensions ready for use were cooled on ice in 5mL  polystyrene tubes (Falcon #2054) for 20 minutes to minimize damage caused by freezing and to prevent repair.  2.2  Irradiation and Dosimetry Cells were irradiated as 1.5mL volumes on ice in a Plexiglas jig with a removable top  in which 4 holes were bored to accommodate the 5mL sample tubes. A 250 kVp Xray unit  Materials and Methods 28 with a 0.5mm copper filter, provided a dose rate of 7.9 Gy/min. Dosimetry for the jig was established under conditions identical to those of a typical experiment using a Victoreen Model 500 electrometer. Measurements were carried out using a probe of 0.6cc volume which was fitted tightly with a rubber balloon throughout measurement s so as to prevent damage caused by immersion in PBS. A l l measurements were taken at 4°C using 1.5mL PBS filled polystyrene tubes surrounded by ice to be consistent with radiation experiments. Results obtained using water filled jigs deviated from those filled with ice due to scattering phenomenon associated with the interaction of X-rays with ice crystals (data not shown). Each measurement for the time course was done three times for each jig hole and for three angles of tube entry (to account for possible handling inconsistencies) and averaged. Dosimetry was calculated using the following formula:  D = (Electrometer  reading) x ~%93 x  7  6  % x 1.07737  Corrections to standard temperature and pressure are taken into account.. The six digit numerical value includes conversion of exposure dose in roentgens to absorbed dose in rads as well as a calibration factor for the electrometer.  A l l experiments were conducted at  766mm and 277K. Table 2. land Fig.2.1 provide the best representation of the data.  Table 2.1. Dosimetry of 250kV, 0.5mm Cu Filter X-ray Unit Time (min) Calculated Dose ((Jy) Standard Error (Gy) 0.00 0.00 0.0 0.025 0.05 0.379 0.047 0.1 0.759 0.2 1.52 0.091 0.170 0.4 3.05 3.82 0.238 0.5 0.390 5.77 0.75 0.574 7.68 1.0 0.522 1.2 9.23 Values shown are the mean and standard error for 3 independent measurements.  Materials and Methods 29  10  0.0  0.2  0.4  0.6  0.8  1.0  Time (minutes) Dose Rate calculated from slope is 7.69 (0.43) Gy/min  Figure 2.1: Dosimetry of 250kV, 0.5mm C u Filter Using Victoreen Model 500 Electrometer.  1.2  Materials and Methods 30  2.3  Comet Assay Technique  2.3.1  Preparation of Slides A  concentration on average  of 60,000 cells/mL was obtained by quickly  micropipetting 100|lL aliquots from 1.5mL initial suspension into 400|J,L ice cold PBS. This step prevented post-irradiation repair in conventional radiation comet assays and was carried over to enzyme experiments for comparison purposes and to eliminate temperature fluctuations.  Cell suspensions were combined with 1% low temperature gelling agarose  (Sigma type VII) in a 3:1 ratio, thoroughly mixed by pipette and spread uniformly onto fully frosted microscope slides (Fisher Finest). Preparation of slides was done in a random sequential fashion to avoid systematic error, and timing between gelling and lysis steps was kept consistent. Slides were precoated with 100|iL of agarose to ensure adhesion of gels to surface throughout incubation stages. This has proven essential when increased handling is necessary as well as when using higher temperatures such as required for neutral lysis. Gels of 1.5mL volume were usually used in radiation assays, however, thinner gels were needed for enzyme work to improve homogeneity by allowing uniform diffusion through agarose. Gels of l m L volume with resulting 2mm thickness were found to be optimal for analysis. Slides were allowed to gel thoroughly (2-4mins) at room temperature to prevent erosion of the top surface which has been found to result when 'leaky gels' are incubated in detergent based solutions. To prevent repair, gelling speed was increased by placing the slides on a glass surface maintained on ice, a provision being that they were removed in less than 1 minute to prevent damage, changes in conformation and diffusion problems brought on by freezing. Typical controls prepared using this method did not vary from those prepared at room temperature.  Materials and Methods 31  2.3.2  Alkaline Lysis Incubation of slides for 1 hour in 1.2M NaCl and 0.03M NaOH in 400mL volumes in  covered pyrex containers lysed cells and removed most proteins.  Experiments aimed at  optimization of lysis time and salt concentration were performed to obtain a homogeneous cell lysis and achieve a salt content that could be rinsed free easily. Although it is known that the histone tetramer H3-H4 does not fully dissociate until a 2 M NaCl concentration is achieved, the presence of these and other nuclear matrix bound proteins did not seem to pose a problem in assessing breaks [Olive et al. 1990], perhaps because these histones, while physically present, do not associate sufficiently closely with D N A after treatment with 1.2 M NaCl. Once pH exceeds about 11.6, D N A begins to denature, and at pH 12.3, the hydrogen bonding between bases is completely disrupted. In this manner, single stranded ends are free to migrate independently of their sister strands. N-laurolyl sarcosine sodium salt (Sarcosyl) at 0.5% was added to the lysis solution to permeabilize membranes  and prevent  D N A damage  by endogenous  nucleases  or  contaminants. Slides were rinsed free of detergent and salt in three successive rinses in large volumes (consistently 400 mL) for a total time of 1 hour in the dark at room temperature. This step is very important since salt has been found to retard migration during electrophoresis as it diffuses out of agarose, leading to images that are difficult to reproduce. Following this rinse step, slides were electrophoresed in fresh buffer of consistent pH to ensure reproducibility and to maintain denaturation. Electrophoresis proceeded at 20V for 25 mins in a 1.8L BioRad model horizontal electrophoresis chamber. A typical current of 60 mA for alkaline conditions fluctuates with increased salt levels and buffer volumes, so care was taken to prepare buffer immediately prior to electrophoresis. The electric field produced  Materials and Methods 32 is fairly uniform resulting in comet images irrespective of position on the slide or location in the chamber [Olive, 1990]. Following electrophoresis, slides were neutralized by quickly rinsing in doubly distilled water and then stained with the fluorescent D N A intercalating dye propidium iodide (2.5 fig/mL) for 30 minutes at room temperature.  Propidium iodide was  chosen based on studies comparing the accuracy of D N A detection and minimization of background for a number of dyes [Olive, 1992].  2.3.3  Neutral Lysis Incubation of slides at 50° for 3 hours in large volumes (400 mL) of 30mM E D T A  and 0.5% SDS pH 8 was required to remove cellular proteins.  Longer lysis times are  necessary for lysis at lower temperatures, while the use of higher temperatures results in breakdown of agarose adhesion to slides and gel loss. Slides were rinsed overnight in T B E (90mM Tris, and 90mM boric acid and 2mM EDTA) and electrophoresed in fresh T B E buffer. D N A staining required up to 45 minutes.  2.3.4. Factors Influencing Migration Technical and biological factors which affect migration include lysis  conditions,  presence of salt, D N A size and packaging, replicating structures and crosslinks [Olive et al.., 1992].  The effect of E D T A was also examined since it appears to affect chromatin  conformation [Wlodek et al.., 1992].  2.3.5  Comet Collection Individual comets were viewed using a Zeiss epifluorescence microscope attached to  an intensified solid state C C D camera (Optikon) and image analysis system. For viewing propidium iodide stained cells, illumination was performed with green light (546nm) excitation from a 100W mercury light. Emission was monitored using a 580 nm reflector and  Materials and Methods 33 a 590 nm barrier filter. Images appearing on the monitor were assigned multicolours that accorded with pixel intensity. This system allows comets to be collected in real time, so 200 comets can be collected, digitized and stored in approximately 20 minutes. Only overlapping images or obvious debris were excluded from collection, ensuring maximal objectivity.  2.3.6  Comet Analysis Using software written by Dr. Ralph Durand of the B C C R C , bivariate plots of the  data can be generated for detailed analysis. The plots, depicted in the results section, not only can be interpreted on a single cell response level, but in addition, given the skew and shape of the distributions, hypoxic fractions, cell cycle effects and crosslinks can be assessed. Three endpoints were used to quantify D N A damage: D N A content, tail moment and percentage of D N A in the tail. The most informative feature is considered to be tail moment, which best correlates with results [Olive et al. 1990], however, D N A content is useful in determining loss of D N A caused by heavy damage to strands; fragments of such small size (<40kb) are pulled from the field of view of the comet or they diffuse out during lysis and rinsing.  This  endpoint is especially useful for correlating restriction enzyme cutting  frequency with radiation induced damage. Percent in tail is defined simply as the fraction of total D N A which migrates relative to the undamaged 'head', while tail moment incorporates both a measure of breaks and tail length. The tail moment is the product of the percentage of D N A in the tail distribution and the mean displacement between the head and tail. It is calculated from pixel intensities and is background corrected [Olive et al., 1990]. A l l three endpoints are expressed in arbitrary units. Damage is profiled relative to control points.  Materials and Methods 34  Tail moment = % DNA in tail x distance between means of head and tail distributions  - i f i i p p i j i i  Fig. 2.2: Digitized images of 2 comets in the same field, showing differences in D N A damage. Direction of D N A migration in the electric field is to the left. Note that as the amount of D N A in the tail increases, the intensity of fluorescence in the head decreases. 2.4  EcoRl Treatment of Cells Cells were cultured and handled as previously described in general methods. Thin  gels (1 mL) were generally used unless otherwise stated. Slides were incubated for 20 mins on ice in 1% TX-100 to permeabilize cells and then were either further incubated for 30 mins at room temp in 2 m M NaCl/ 2mM Na EDTA to remove cellular proteins, followed by 2  equilibration in reaction buffer (3x5mins) or they were directly equilibrated to remove detergent, E D T A and excess salt. Unless otherwise stated, all volumes used were 400 mL and containers were covered pyrex dishes, extensively cleaned of extracellular nucleases prior to the experiment. EcoRl (Gibco) was applied by first diluting the original stock (10 U/(xL) to the appropriate dosage with reaction buffer in autoclaved ependorf tubes [BDH, Vancouver] before micropipetting 100 | i L directly onto agarose. A single unit (U) of enzyme is equal to  Materials and Methods 35 the concentration required to completely digest ^-bacteriophage D N A in 1 hour at optimal reaction temperature. Care was taken to spread enzyme evenly before covering with cleaned coverslips and incubating for 45 mins at 37°C in pre-warmed incubation chambers. Neutral lysis, rinse in T B E overnight, electrophoresis, and analysis steps were as previously described NaCl concentrations used for altering chromatin structure ranged from OM to 3 M and the dose range for R E ranged from 0 to 10 U/|iL. Enzyme was thawed immediately prior to application and maintained on ice throughout to ensure minimal loss of activity. A l l data shown used the same enzyme stock. temporize physiological conditions.  Some experiments used PBS instead of water to  Most experiments involved an initial lysis stage on ice  in order to compare with radiation-induced breaks. Great care was taken in all experiments to avoid contamination as well as to ensure consistency of timing, temerature and pH. Two radiation experiments were performed in conjunction with restriction enzymes in order to see if conformational changes induced by X-rays could be assessed.  The first  experiment allowed a 1 hour repair to follow 15Gy irradiation, in order to test for relative ability to regain original conformation. The second examined the immediate conformational changes resulting from 2Gy.  2.5  Detection of Base Damage Using the Comet Assay V79 cells were cultured and handled as described earlier (Section 2.1). Slides were  covered with l m L of 3:1 agarose unless otherwise stated to overcome diffusion problems. Cells were lysed using a number of solutions, summarized in 3.11 to reduce background breaks and heterogeneity. Lysis was either at room temperature or at 4°C; pH was adjusted to 10 for some experiments. Slides were equilibrated in reaction buffer specific for the enzyme  Materials and Methods 36  3:1 Agarose  Permeabilization  Single cell suspension  Apply enzyme; cover  Equilibration  Hisones removed  Incubate  Neutral Lysis  Analysis Electrophoresis, P.I. Staining Rinse  Figure 2.3: Schematic of EcoRlcomet assay protocol. Details are described in Materials and Methods, section 2.4.  Materials and Methods 37 (Table 2.2) with various levels of E D T A in order to reduce extracellular nuclease activity. Optimum combinations of enzyme concentration and E D T A level in buffer were sought using two of the lysis methods. Experiments were mostly done in the dark, as it has been reported that base damage can be caused by visible light [Pfaum, et al., 1994]. However, as no difference was observed between such experiments and those where cells were briefly exposed to light during transfers, no attempt was made to consistently achieve this condition.  Table 2.2: Description of Buffers for Enzymes Used to Detect Base Damage Enzyme  Recipe  Temp  PH  7-endonuclease fraction I from m.luteus (crude extract) 7-endonuclease fraction II Endonuclease III (lug/mL)  4()mM NaCl, 3()mM Tris. 1 mM Na2EDTA, pH adjusted by addition of NaOH  37 °C  7.6  same as above  same  same  40mM Hepes, 0.1M KC1, 0.5mM Na2EDTA, 0.2mg/mL B S A ; pH adjusted by addition of K O H  37°C  8.0  Application of enzyme was done using three methods in order to compare efficiency of diffusion and to develop a method which required less enzyme, to reduce expense. A l l incubations were performed for 20, 25 or 30 mins at 37°C using either a crude extract isolated from M. luteus (fraction I achieved following recipe of Jorgensen, 1987), or a more purified version, obtained from Dr. T. Jorgensen at Georgetown University. The activity of M . luteus extracts was not assessed. However, by personal correspondence, Dr. Jorgensen conveyed that on average, 1800 U/mg of specific activity resulted using this extract. In addition, endonuclease HI was generously supplied by Dr. S. Wallace at the State University of New York.  Materials and Methods 38 Since lOOfiL of buffer (with or without addition of enzyme) was the most frequently used technique, crude extract enzyme concentrations are expressed as a dilution factor multiplied by 100; for example when 10|iL of extract was diluted into 90|xL buffer prior to application, this is noted as a concentration of 1:10, or simply 10. Following incubation with enzyme, cells were lysed, rinsed, electrophoresed and stained and analyzed according to the previously described alkali comet technique. The ratio of enzyme sensitive sites to SSBs (Ness/Nssb) is calculated as follows:  Ness/Nssb ={(M.luteus+SSB) - SSB}/(SSB)} where SSB = strand breaks produced by radiation, and M . luteus + SSB refers to total number of strand breaks after cells are treated with enzyme. Values are obtained from mean tail moments.  Given the amount of background damage (ie: damage detected in unirradiated  treated controls) the ratio is calculated from individual measurements instead of slopes.  2.6 Statistical Analysis The significance of the difference in means between two independent samples was determined using the t-Test in addition to performing one-way analysis of variance ( A N O V A ) on a point-by-point basis. Curve fitting was not practical since it is uncertain whether or not enzyme dose responses are linear; F and t values correspond to differences in means of individual dose points. Values of Fcrit. and tcrit. appear in Appendix 1. Standard deviations for histogram (Gaussian) distributions were generated by comet software based on the formula:  Materials and Methods 39 For standard error calculations of three experiments, the N in the denominator is replaced with N - l . The numerical difference between the two equation forms is virtually insignificant [ Taylor, 1982]. P values for probabilities under 0.1, 1 and 5 % were obtained from statistical tables based on degrees of freedom for each set of data. These values are appended to tables where A N O V A and t Test statistics were performed and the number of experiments for each set is indicated. For A N O V A analysis, two degrees of freedom are specified which incorporate the degree of variability within groups (dfw) and also differences between groups (dfb). For t Test statistics, the total number of degrees of freedom (df) is used. These values are related to the total number of data points (N) and the number of groups (k) as follows:  dfw = k-1; dfb = N-k; df = N-2 For this study, the number of groups used for comparison is 2 (TK6 and V79 cells) so the dfb is always equal to one, however some measurements were taken more than three times for a cell line, which changed the value of dfw. For a detailed reference of formulas used, refer to Kranzler and Moursund, 1995. Propagation of errors for independent, random uncertainties was calculated using:  For experiments where fewer than three independent experiments were done, the number of comets collected is shown in addition to standard deviations. For independent experiments performed three times, 100-300 comets were collected.  Results 40 3.  RESULTS  3.1  X-Ray-Induced DNA Damage Detected in V79 and TK6 Cell Lines Using the Alkaline and Neutral Comet Assay. Figure 3.1 shows the previously determined [Olive et al. 1991 and Olive et al. 1996]  clonogenic survival response of Chinese hamster V79 cells and human lymphoblastoid T K 6 cells to ionizing radiation. Table 3.3 summarizes the data. Clearly the V79 cells are much more resistant to killing than the TK6 cells. For this reason alone, these two cell lines were chosen for further studies to address the question of whether higher order chromatin structure could explain, at least in part, the differences in intrinsic radiosensitivity of these two cell lines. In particular, would it be possible to detect differences in D N A damage by ionizing radiation, or differences in the accessibility of chromatin to REs or base repair enzymes? Figure 2.2 shows the typical appearance of comets in the neutral comet assay. Results in Fig. 3.1 and in Tables 3.1 and 3.2 indicate that the response to ionizing radiation, in terms of initial SSB and DSB detected, was identical for these two cell lines. Table 3.1: X-ray-Induced SSBs Detected by the Alkaline Comet Assay V79 Cells TK6 Cells MTM % in Jail % in Tail DNA Content Dose(Gy) DNA Content! MTM 6.3 ± 1.4 1.1 ± 0 . 5 20.1 ± 6 . 2 8.9 ± 2.2 1.1 ± 0 . 9 22.8±13.2 0.0 2.3 ± 1.2 31.0 ± 14.7 8.4 ± 2 . 5 3.0 ± 1.8 32.3 ± 3 . 8 6.6 ± 1.9 1.0 1.2 4.9 ± 0 . 3 1.5 ± 0 . 5 20.1 ± 5 . 9 6.9 ± 2 . 1 2.4 ± 1.4 27.1 ± 9 . 9 7.7 ± 2 . 0 4.3 ± 1.5 38.5 ±10.3 2.0 2.5 4.6 ± 0 . 6 3.1 ± 0 . 7 24.5 ± 6.9 4.4 ± 0.4 3.75 6.1 ± 1.7 41.4 ± 4 . 4 9.3 ± 2 . 1 54.6 ± 8 . 1 8.3 ± 2 . 1 53.6 ± 9 . 3 7.2 ± 2 . 1 5.4 ± 1.7 4.0 8.3 ± 2 . 1 54.1 ± 1.7 5.0 4.0 ± 0.5 11.2 ± 2 . 3 61.6 ± 8 . 0 6.4 ± 1.8 8.7 ± 1.7 53.5 ± 8 . 1 6.0 5.6 ± 1.8 62.8 ± 4 . 5 10.3 ± 1 . 8 6.2 4.0 ± 0.4 13.6 ± 2 . 4 70.7 ± 8.2 5.8 ± 1.6 5.2 ± 1.9 9.9 ± 1.5 60.7 ± 7.7 8.0 15.0 ± 1.7 75.2 ± 5.4 6.0 ± 1.7 10.0 5.0 ± 2 . 0 14.4 ± 1.8 71.9 ± 5 . 5 18.1 ± 2 . 4 85.1 ± 4 . 2 5.5 ± 1.7 3.9 ± 1.4 16.5 ± 2.0 82.2 ± 4 . 7 15.0 Values shown are the means and standard errors for 3 independent experiments. (10C comets were scored. A l l endpoints of D N A damage are expressed in arbitrary units.  Results 41 Table 3.2 : X-ray-Induced Double Strand Breaks Detected by the Neutral Comet Assay V79 Cells TK6 Cells DNA % in Tail # DN\ MTM % in Tail # MTM Content Content 1.7 ± 0 . 8 0 28.5 ± 13.7 130 8.2 ± 2 . 2 i . 2 ± o . y 23.3 ± 16. ) 105 7.2 ± 2 . 2 6.6 ± 2 . 4 1.8 ± 0 . 7 31.8 ± 13.9 58 8.5 ± 2 . 2 2.2 ± 1.1 27.5 ± 14.7 62 1 7.5 ± 2 . 2 1.6 ± 0 . 9 24.6 ± 13.6 108 9.0 ± 2 . 3 1.2 ± 0 . 7 19.2 ± 11.6 110 2 7.4 ± 2.7 2.2 ± 2 . 1 32.9 ± 18.1 108 8.9 ± 2 . 2 1.4 ± 0 . 9 19.0± 11.0 104 9.1+2.4 2.4 ± 1.0 29.8+ 16.1 59 4 8.1 ± 2 . 4 2.5 ± 1.2 34.7 ± 16.6 100 9.0± 2.5 1.7 ± 1.0 27.7 ± 16.3 136 6 8.0 ± 2 . 3 2.5 ± 1.2 34.9 ± 16.9 111 9.5 ± 2 . 4 2.5 ± 2 . 1 32.4 ± 18.6 112 8 7.5 ± 2.0 2.6 ± 2.0 34.4 ± 16.9 110 9.6 ± 2 . 6 3.2 ± 2.7 35.7 ± 19.5 118 7.7 ± 2.7 3.1 ± 1.3 34.3 ± 12.8 100 9.2 ± 2 . 7 3.2 ± 1.2 35.7 ± 14.6 63 10 7.9 ± 2.4 3.1 ± 1.3 31.6 ± 11.0 61 9.7 ± 2 . 6 2.9 ± 1.7 33.4 ± 16.5 122 15 7.2 ± 2.4 3.0± 1.2 35.4 ± 10.7 110 9.3 ± 2 . 7 3.5 ± 4 . 6 32.5 ± 12.7 140 7.4 ± 2 . 6 4.6 ± 1.5 42.9 ± 11.6 56 9.8 ± 2 . 6 3.9 ± 1.5 37.2 ± 11.3 58 1  Values shown are means and standard deviations for one or two measurements (where two values are given). Standard deviations are obtained from histograms generated by comet software. Number of comets scored is shown in right hand columns.  Table 3.3: Clonogenic Survival Data for V79 and TK6 Cell Lines Damaged by X-rays  Dose (Gy) 0 1 2 3 4 5 6 8 10 12 14  V79 Cells Surviving Standard Fraction Error 1.00 0.73  0.06  0.48  0.06  0.28 0.12 0.046 0.012 0.0018  0.046 0.03 0.017 0.006 0.0013  TK6 Cells Surviving Standard Fraction Error 0.001 1.00 0.14 0.33 0.05 0.11 0.004 0.019 0.001 0.003 0.0003 0.0006  Data reprinted with permission from Olive et al. [1991] and Olive et al. [1996].  Results 42  DNA Damage Profiles  Clonogenicity  O V79SSB • TK6SSB • V79DSB • TK6DSB  O V79 Cells • TK6 Cells  q  o co LL  U) c >  00  108  0.01 h  r  75 0.001 h  'CO  — I  _c  50 25 -  0 -  0  5  10  Dose (Gy)  15  20  0.0001  0  5  10  15  Dose (Gy)  Figure 3.1: Comparison of DNA Damage Induced by X-rays with Clonogenic Survival Curves for Chinese Hamster V79 and Human Lymphoblastoid TK6 (radiosensitive) Cell Lines. All damage endpoints are expressed in arbitrary units.  20  Results 43 3.2  EcoRl Induced DNA Damage in V79 and TK6 Cells Using the Comet Assay Table 3.3 and Fig. 3.2 show results for EcoRl-induced D N A damage in V79 and TK6  cells. Cells were exposed to the restriction enzyme after removal of histones with NaCl and after permeabilization with 1% TX-100 either in doubly distilled water or PBS. As expected for histone-free D N A , there was no appreciable difference using either diluent, however, for intact structures, there appeared to be a loosening of chromatin in PBS. For example, at an enzyme concentration of 0.1U/p:L, the ratio of MTM.s was about 2-fold for PBS vs H 0 . 2  Table 3.4: Effect of Lysis Solvent on Accessibility to EcoRl for V79 Cells Lysis: 2M NaCl/2mM Na2EDT A/0.5% TX-100 45 mins incubation Solvent # MTM % in Tail Dose(LVuL) DNAContent PBS 0 100 37 8 ± 12 7 8.9±3.7 3.7 ± 2 8 250 8.0±3.2 2.8 ± 4 . 5 30.7 ± 13.5 0.01 150 6.2+ 2.4 29.2 ±7.4 83.4 ± 10.9 100 4.4+2.1 30.1±7.2 90.9± 6.5 36.6±2.4 H0 0 6.9±0.2 3.5±0.3 32.2±3.4 85.6±2.4 0.01 5.6± 1.6 Lvsis: 0.5% TX-100/2mM Na EDTA 1 hour incubation 200 PBS 0 5.2 ± 3 . 7 2.5 ± 1.5 33.9 ±12.0 100 1.6± 0.7 28.7 ± 8.7 5.2 ± 1.6 100 0.001 4.4 ± 1.1 1.4± 0.61 25.2 ± 8 . 5 200 2.8± 1.5 35.4± 12.5 4.6± 2.3 100 0.01 27.7 ± 11.9 5.9± 1.7 2.1± 1.8 202 4.4± 3.0 4.5 ± 4.4 38.1 ± 13.8 100 0.01 4.7± 1.5 5.1 ± 5 . 8 38.7 ± 16.5 18.3 ± 12.5 67.5 ± 23.0 100 0.1 4.0 ± 2 . 0 83.2 ±18.7 100 0.2 3.6 ± 2 . 7 27.2±10.3 34.4±1.4 3.3± 0.2 H0 0 8.1±0.6 38.3±0.7 7.9±0.6 3.8±0.2 0.001 3.7±0.3 35.9+1.8 0.01 8.1+1.0 37.7±0.4 0.01 6.9±0.8 4.2±0.3 9.5±2.4 50.1±4.5 0.1 7.4±0.9 69.3±1.4 19.9+1.4 6.9±0.8 1.0 Values shown for PBS as solvent are means and standard deviations for one or two measurements. Values shown for H 0 as solvent are means and standard errors for three independent experiments. 2  2  2  2  Results 44  0.5%TX-100/1.2M NaCl/ 2mM EDTA  0.5% TX-100/1 mM EDTA  c CD c o O  < Q  c CD £ o CO  h-  c co  CD  CO  — I  c  CD  O i_  CD  Q_ 0.01 Dose (U/uL)  0.01 Dose (U/uL)  Figure 3.2: Effect of Solvent Used in Lysis Solutions  1.0  Results 45 To address the issue of large scale conformational changes induced by ionizing radiation, radiation experiments were conducted in conjunction with enzyme application and are summarized in Tables 3.5 and 3.6, Figs. 3.3 and 3.4. For the enzyme dose response curves using irradiation with 15 Gy and consecutive 1 hour repair, and for 2 Gy and no repair, the extent of enzyme cleavage was virtually identical in both cell lines. Interestingly, though, TK6 irradiated controls in the latter experiment (ie: incubated with buffer only) showed an increase in damage over cells lysed immediately after irradiation such as assessed previously in regular neutral comet assays (one measurement only; highlighted in Table 3.6). Table 3.2 shows mean tail moment increases (unirradiated control subtracted) for V79 and T K 6 cells respectively of 0.5±2.2 and 0.2±1.3 for a 2 Gy dose, while cells lysed in salt and detergent, incubated for 45 minutes at 37°C prior to neutral lysis show mean tail moments of 2.3±2.1 and 10.7±8.8 (Table 3.6). Although the error is very large, due to heterogeneity effects, over 70% of individual T K 6 cells show a tail moment greater than 10, which is the upper limit observed in V79 cells. Bivariate plots of tail moment versus D N A content for these observations are shown in Fig. 3.5.  Table 3.5: Dose Response for EcoRl Following 15Gy Irradiation and 1 hour Repair  Dose UAtI,  DNA Content  0 0.01 0.1 1.0  7.2±1.9 7.7±2.3 7.0±2.3 6.7±2.5  V79 Cells MTM % in Tail 3.1±1.3 3.7±3.0 6.8±7.2 21.7±12. 2  33.6*10.3 34.8±12.8 41.7±16.5 68.0 ±19.7  #  DNA Content  94 111 98 150  8.9±2.2 8.7±7.2 9.2±2.8 8.7±2.5  TK6 Cells MTM ' in Tail c  f  4 5± 1 6 41.8±10.2 4.4±1.5 40.9±8.8 7.4±3.8 47.2±12.8 16.6±8.6 61.5+15.7  Values shown are means and standard deviations for one measurement only. Standard deviation was obtained from histograms generated by comet software.  # 99 95 93 96  Figure 3.3: Dose Response Using E c o R l Following 15Gy and 1 Hour Repair for V79 and TK6 Cell Lines.  Results 47  Table 3.6: Effect of Salt Concentration Using O.OlU/uX EcoRl Following 2Gy Irradiation  Dose NaCl DNA LVfiL mM Content 0  0.01  0.1 0.2 0.4 1.2 0.1 0.2 0.4 1.2  V79 Cells MTM % inTail #  7.1±2.1 2.3+0.9 7.9±2.3 2.4+1.1 6.7±2.1 3.0±1.2 5.9±1.8 4.6±1.9 6.7±2.0 2.8±3.8 6.6±1.8 2.6±1.4 6.1±2.1 9.9±7.3 5.2±2.2 27.5+9.0  3()6±1 1 6 160 29.0+11.1 164 33.3±10.5 110 43.3±8.5 161 31.5+13.1 162 30.8±10.6 210 51.4±19.7 229 83.0+16.6 194  DNA Content  TK6 Cells MTM <3 inTail #  8.7±2.4 4 4+1.7 4o.:±i i o 155 8.7±2.6 4.1±1.4 37.7±10.6 160 7.9±2.4 3.8+1.5 37.3±9.0 121 7.2±2.4 15.1±8.6 62.3±18.0 159 8.2±2.4 5.1±6.0 40.5±14.7 181 8.1±2.4 3.8±4.0 37.0±11.8 155 7.4±2.4 8.8±7.6 50.0±16.4 213 6.8±2.6 31.6+8.4 86.4±11.7 169  Values shown are means and standard deviations for one measurement only. Standard deviations were obtained from histograms generated by comet software. Dose response curves obtained using permeabilizing or 1.2M NaCl lysis conditions for 3 independent experiments show no significant difference in terms of accessibility (see F and t values in right hand columns of Tables 3.7 and 3.8), although it is interesting that the tail moment decrease for TK6 cells at O.OlU/jxL enzyme concentrations, using high salt, is accompanied by more severe D N A loss. (Table 3.9 and Fig 3.7a). Points are within error range, however, it is worth noting the trend towards a more rapid decline in loss of TK6 cells, which was consistent amongst several experiments. In contrast, practically no D N A was lost when using 1% TX-100 conditions and there is correspondingly little variation in extent of damage (Table 3.9 and Fig. 3.7b). In addition, the error was much smaller (<25% for highest error), reflective of homogeneity of results in individual experiments and also the reproducibility of the assay.  Results 48  Figure 3.4: Effect of Salt Concentration Using 0.01 U/uL E c o R l Following 2Gy Irradiation for V79 and TK6 Cells.  Results 49  Neutral Lysis Only  Lysis in 0.5% Tx/2mM EDTA/1.2MNaCI Incubation @37°Cwith Buffer, Neutral Lysis.  V79  TK6  0  10 DNA Content  0  10  20  DNA Content  Figure 3.5: Bivariate Plots Comparing the Response to an X-ray Dose of 2 Gy of V79 and TK6 Cells Lysed Under Two Different Conditions.  Results 50  Table 3.7: EcoRl Dose Response for Cells Permeabilized by 1% TX-100/2mM Na EDTA 2  V79 Cells Dose DNA U/|iL Content 0.0 0.001 0.01 0.01 0.1 1.0 10.0  8.1 7.9 8.1 6.9 7.4 6.9 7.0  ±0.0 ±0.7 ±1.0 ±0.8 ±0.9 ±0.8 ±0.8  MTM 3.3±0.2 3.8 ±0.2 3.7 ±0.3 4.2 ±0.3 9.5 ±2.4 19.9+1.4 37.2 ±1.2  TK6 Cells "r in Tail  DNA Content  34.4 38.3 35.9 37.7 50.1 69,3 91.6  7.8 8.2 8.4 8.4 8.1 7.5 6.9  ±1.4 ±0.7 ±1.8 ±0.4 ±4.5 ±1.4 ±0.6  ±0.4 ±0.4 ±0.3 ±0.5 ±0.4 ±0.3 ±0.7  MTM 3.8 ±0.5 3.6 ±0.5 4.0 ±0.4 4.3 ±0.3 8.3 ±1.5 17.1+0.9 35.2 ±0.9  MTM Statistics % in Tail F obt. t obt. ttest 37.2 ±2.1 36.4 ±2.4 38.1 ±2.0 37.9±2.0 48.0 ±1.6 60.8±1.2 85.6 ±0.6  0.29 0.00 0.42 0.00 0.22 3.14 1.89  0.63 0.31 0.52 0.19 0.45 1.78 1.35  Values shown are means and standard errors for three (V79) and 5 (TK6) independent experiments. Fcrit and t crit values are shown in Appendix.  Table 3.8: EcoRl Dose Response for Cells Lysed in 1.2M NaCl Prior to Incubation V79 Cells Dose I ) \ \ U/uX Content 0.0 0.0001 0.001 0.01* 0.01* 0.1**  7.6 ±0.9 7.7±0.8 7.6 ±1.0 7.2 ±1.0 6.6 ±1.0 3.7 ±0.9  .MTM Statistics k in Tail V obt t obt t test  TKACi-lls  MTM  % in Tail  I)N\ Content  MTM  4 7 ±0.1 4.7 ±0.3 5.8 ±0.8 15.6 ±4.5 28.9 ±1.2 37.0 ±3.7  42 3 ±1.0 43.7 ±0.2 44.4 ±1.0 61.2 ±5.0 82.4 ±1.8 87.9 ±4.1  7.9 ±0.6 8.0 ±0.8 7.4 ±0.8 6.9±1.0 5.6 ±1.2 2.8 ±0.6  4.0 ±0.5 4.2 ±0.6 5.7 ±1.0 14.7± 3.2 26.8 ±4.2 32.4 ±3.2  c  38 0 ±2 6 38.1 ±2.4 42.2 ±2.9 57.9 ±5.8 73.6 ±6.4 84.5 ±3.5  0.92 0.36 0.00 0.03 0.20 1.18  1.04 0.60 0.07 0.42 0.38 0.70  Values shown are means and standard errors for 3 (V79) or 5 (TK6) independent experiments. * Refers to 4 (V79) and 5 (TK6) independent experiments ** Refers to 4 (V79) and 3 (TK6) independent experiments. Fcrit. and tcrit. values are shown in Appendix.  Results 51  Figure 3.6: Dose Response Curves Using E c o R l for V79 and TK6 Cell Lines Under Low and High Salt Lysis Conditions.  Results 52  Table 3.9: Comparison of V79 and TK6 Cell Lines: Correlations Between MTM Decrease and DNA Loss for Cells Lysed with 1.2M NaCl Prior to Enzyme Treatment.  Dose DNA U/uL (out/ 0  9.4±2.8 7.0±2.6 6.3±2.2  V79 Cells MTM Fr. of DNA Lost 4.8± 3.2 4.4+ 1.5 4.8±2.9  A MTM  #  DNA ('out.  TK6 Cells Fr. of MTM A MTM DNA Lost  82 7.3+2.3 125 9.4± 3.0 176 6.0+ 1.9 8.9±2.4  0.01 8.6±2.9 0.1+0.5 27.0+10.4 22.2±10.9 72 9.1± 3.7 6.2±2.3 0.1+0.4 31.1± 9.0 26.6±9.1 113 7.9± 2.5 4.5±2.5 0.3±0.6 28.4±10.4 23.6±10.8 151 3.7±2.1 3.0± 2.4  4 3± 4.7± 3.7± 5.1+ 0.0±2.4 0.2±0.3 0.4±0.5 0.7±0.4  124 150 200 300  1 6 1.8 1.5 1.6  35.7±10.3 30.5±10.3 15.1±10.2 18.5±12.5  #  31.4±10.4 25.8±10.5 11.4±10.3 13.4+12.6  200 192 130 69  Values shown are means and standard deviations (obtained from histogram generated by comet software) for three independent experiments to show consistent correlation of D N A content loss with Mean Tail Moment decrease (MTMD) for TK6 and V79 cell lines.  The effect of salt on cells damaged with 0.01 U/jiL EcoRl also yielded little information about possible differences between cell lines; the point at 1.2M NaCl deviated from the otherwise merging lines, however, the dose response curves previously shown contradict this data indicating that it may be an anomaly (Figs. 3.6b and 3.8). The F value of 4.44 and t value of 1.6 are well below significance. The initial 'dip' for concentrations between 0 and 0.2M NaCl is most likely due to the electrostatic nature of the chromatin, which reverts to a more compact form at increased cation concentrations; however, it is also possible that it is in part a result of staining properties.  Results 53  (a) 1.2 o c  o  c o O  1.0  i  p  i  1  i  i 1  0.8  i  J!  n Low Salt Lysis  0.6 0.4 -  <  0.2  Q  0.0  • •  V79 TK6 i  1.2 o  1.0  c c o O  B  -] High Salt Lysis  < Q  10.0  CD CO CO CD  o CD Q  J  -+—<  c:  High Salt Lysis  CD  CO  I-  c co  CD  1.0 Fraction of DNA Lost Figure 3.7: (a) Comparison of DNA loss for V79 and TK6 Cells as a Function of Enzyme Dose, (b) Correlation Between Mean Tail Moment Decrease (control subtracted) and DNA Content Lost for Both Cell Lines Using High Salt Lysis Conditions, fr.c is fraction of control.  Results 54  Table 3.10: Effect of Salt Using O.OlU/uX EcoRl V79 Cells NaCl (M) 0.0 0.1 0.2 0.4 0.6 0.8 1.0 1.2 2.0  # 4 4 3 3 3 3 2 3 3  DNA Content 6.9±0.2 6.6±0.2 6.9 ±0.3 6.4 ±0.6 6.6 ±0.8 6.1 ±1.0 5.0 ±1.3 6.2 ±0.9 5.6 ±1.6  MTM 3.4±0.3 3.1 ±0.2 2.8 ±0.3 9.6 ±2.2 13.6 ±2.8 19.8 ±4.5 27.6 ±1.1 31.2 ±3.0 32.2± 3.4  Tl<r> evils <;i in Tail # 36 6 ±2 4 33.1 ±0.2 32.4 ±1.9 49.2 ±3.5 55.4 ±6.0 65.2±5.9 76.9 ±2.4 84.0 ±1.0 85.6 ±2.4  4 4 5 3 3 4 2 2 3  DNA Con:cnt 7.9±0.4 8.0 ±0.1 8.4 ±0.1 8.4 ±0.2 7.9 ±0.6 7.6 ±1.6 5.8 ±2.0 7.0 ±0.3 7.6 ±0.3  MTM  % in Tail  4 2 ±0.9 3.6 ±0.4 4.0 ±0.7 9.1 ±2.2 13.4 ±6.7 20.0 ±5.6 30.4 ±4.0 23.0 ±1.8 30.0 ±2.6  39.4 ±3.y 35.4 ±2.8 37.7±4.7 49.4 ±3.4 55.4±10.9 68.2±8.6 81.9 ±3.5 74.0 ±3.9 82.1 ±2.6  MTM Statistics Fohl I ont ttest 0 75 1.7ft 0.98 0.97 5.53 1.11 0.04 0.10 0.003 0.02 0.00 0.02 0.48 1.31 4.44 1.60 0.33 0.42  Fcrit. and t crit. values are shown in Appendix 1. In all studies, the issue of cell cycle-dependent damage was examined by comparing D N A damage with D N A content in bivariate plots (eg. Fig. 3.5). There appeared to be no difference in any of the experiments in terms of response according to position in the cell cycle; damage was uniform for any dose given. For this reason, statistical analysis was not performed.  3.3 Base Damage Detection Using the Comet Assay 3.3.1 Background Damage As indicated in the introduction, there are reports that the comet assay can be used, in conjunction with base damage recognition enzymes, to identify numbers of base lesions present in individual cells [Collins et al., 1995]. However, background damage is often a problem, since crude enzyme extracts can contain exonucleases. Gamma endonuclease is fortunately less sensitive to the presence of E D T A than other nucleases, so inclusion of 2 m M E D T A will reduce background significantly. Of the lysis solutions used, the most consistently  Figure 3.8: Effect of Salt Concentration on V79 and TK6 Cells Prior to Incubation with 0.01 U/uL E c o R l .  Results 56  60  •  I  1  >  >  <—i  •  • "-•— —r 1  • a. 0 Gy •  40 20  80  CO —  _  z—-i—fc-l .  i  .  .  .  :—i—i • i  i  i  .  rH  .  i  "  I  i — i — i — ' — '  i\ .i  •M—-=—i ^ — I\  : b. 10 Gy  60 40  <  "Z  20  Q  1  —,  1  ,  1  ,  1—1— 1  '  '  i  •  i  •  • 1  • 1  • 1  i •  • •  - c. Normalized  • •  •—>—  1 1  •  '  '  .  -  •  .  V 50  0.0  0.5  1.0  1.5  2.0  NaCl Concentration {M)  Figure 3.9: DNA double-strand breakage measured using the neutral comet assay for cells exposed to various salt concentrations prior to irradiation. Panels a and b show the response of unirradiated cells and cells irradiated with 10 Gy: (O) HT144, (•) DU145, (V) U87 and (T) HT29 cells. The mean and standard deviation for three or more experiments are given. In panel c, results in panel b have been normalized  Results 57 low in background damage to untreated controls and heterogeneity was 0.5% TX-100, 1.2M NaCl and 2mM N a E D T A (#9 shown in Table 3.11), however, Collin's lysis (#4) and a 2  modified version using lower levels of salt and E D T A (#5) displayed improved homogeneity of data after enzyme application. A l l three methods were used in conjunction with different amounts of the nuclease inhibitor and chelating agent, E D T A , in reaction buffer, with different enzyme concentrations. V79 cells were used in all base damage experiments.  Table 3.11 #  Influence of Lysis Solution on Background DNA damage Lysis Solution  1 1% TX-100, 2mM EDTA, 0.5% Sure, 1.2M NaCl 2 30mM E D T A , 0.5%Sarc, 1.2MNaCl 3 @pH 10: 4 Collins: 1 %TX-100,lOOmM E D T A , lOmM Tris, 2.5M NaCl, pHIO 5 1%TX-100, 30mM EDTA,10mM Tris, 1.2M NaCl 6 lOmM E D T A , 0.5% Sarc, 1.2M NaCl @ pH 10 7 8 1%TX-100, 0.5%Sarc, 1.2MNaCl 9 0.5%TX-100, 2mM E D T A , 1.2MNaCl 10 1% TX-100, 0.1% Proteinase K  MTM  % in Tail  DNA H Content  #  8.0 ± 4 1 45 9+15 5 7.2±2.4 H 105 5.4 ± 4 . 0 37.6±17.4 7.8±2.3 M 103 3.2±2.9 27.5±16.1 8.3±2.8 L 103 4.9 ±4.2 35.2+17.1 7.4+2.1 M 109 4.2±3.1 31.7±14.3 8.3 ± 2.4 L 4.7±3.7 4.5±3.4 3.6±2.7 1.6+ 1.0 3.4 ± 1.6  35.8±16.8 31.2+15.4 30.1+14.5 17.8+9.7 33.6+13.3  8.4±3.0 7.9±2.5 8.0±2.3 7.2+1.4 6.9+1.5  103  L 103 M 104 L 105 L 60 L 98  Values shown are means and standard deviations for one measurement only. Standard deviation was obtained from histograms generated by comet software. L=Low; M=Moderate; H=High Level of Heterogeneity  3.3.2  Detection of Damage by Micrococcal Endonuclease To address the high level of damage seen in unirradiated treated controls, a control  assay was performed to determine at which stage strand breaks were being formed, (ie: T X 100 incubation, high salt lysis) and slides were consecutively removed from each stage and incubated in alkali lysis. It was determined that minimal damage resulted from extracellular  Results 58 nucleases (<1 in M T M ) and all additional damage resulted upon application of enzyme. In addition, an experiment was performed using the lOmL application technique (see Materials and Methods) in storage buffer alone, to test for damaging effects of 2-f3 Mercaptoethanol, which resulted in undamaged cells (data not shown). To  account for the possibility that the enzyme preparation may have been  contaminated, it was extensively dialyzed and compared, using 5 u i in 1 m M E D T A reaction buffer, to non-dialyzed enzyme extract. The results of one experiment showed tail moments of 13.3 ± 6.6 and 18.3 ± 8.0 respectively a decrease easily achieved using 5mM E D T A in the buffer.  Use of a second purified extract showed no increase in activity over the initial  preparation and the background damage level remained the same (data not shown). For all three lysis methods, there was a compromise between background damage adjustment and base damage detection; overcompensating for background by adding high levels of E D T A to the reaction buffer, inactivated the enzyme as is shown in Fig. 3.15. No definite values for critical concentrations can be assessed, as only 1 or 2 measurements were taken for each point, however, 60mM seemed to be an upper limit; beyond this level, there appeared to be retardation of D N A during electrophoresis, resulting in artificially low damage for irradiated cells. The effect of duration of rinse of high concentrations of both E D T A and NaCl prior to electrophoresis was assessed chiefly by qualitative observations.  For high  concentrations of salt, ranging from 1.2M to 2.5M, a rinse period of at least 40 mins with two changes of buffer was required in order to remove salt. Before this time, V79 cells appeared completely undamaged owing to their inability to migrate during electrophoresis. In the case of E D T A , at levels greater than 30mM, comets improperly rinsed appeared as 'spearheads',  Results 59 thought to be due to non-uniform binding of E D T A to D N A , resulting in some D N A , but not all, able to migrate. The results summarized in Tables 3.14 through to 3.19 show the effect of enzyme concentration on irradiated or unirradiated cells using various levels of N a E D T A . Tables 2  3.14, 3.15 and 3.16 present data obtained using the lysis solution containing l%TX-100/2mM Na EDTA/1.2M NaCl. The corresponding figures (Figs. 3.10, 3.11, 3.12 and 3.17 (endo HI)) 2  illustrate parallel shifting of the dose response curve, as do the results obtained using Collin's lysis and its modified solution. These values are shown, respectively, in Tables 3.16, 3.17 and 3.18; Figs. 3.13, 3.14 and 3.16. The effect of using elevated levels of E D T A in the buffer are summarized for the first and third lysis conditions in Fig. 3.15. A summary of results and values of Ness/Nssb is provided in Table 3.21. Fractions of Ness/Nssb did not vary appreciably among controls, however, it is clear that there is no advantage to using much higher EDTA/enzyme levels and in fact, as it uses more enzyme it is neither practical nor economical to develop this technique. There is definite improvement using lOmM E D T A in the buffer for all lysis methods, presumably because the chelating agent inactivates nonspecific nucleases.  Interestingly, results comparing lOmM Tris-  containing or non-containing lysis solutions show a definite decrease in Ness/Nssb for unirradiated controls, suggestive of either less lysis-induced base damage or more efficient removal of contaminants. The use of Endonuclease HI, further showed little difference using l|i.g/mL and the slope was shifted upwards using an increased concentration of 20 |ig/mL (data not shown). As a crude base damage detection method, results were compared for cells either lysed under normal alkali conditions or at elevated concentrations of NaOH and high  Results 60 temperatures following irradiation. A greater number of lesions in irradiated samples under the extreme lysis was expected as more alkali-labile sites would be converted into breaks, however, an equivalent amount of breaks was found in the controls. This effect, mirroring results using the crude extract, supports the possibility that damage detected using the Micrococcal nuclease could be largely due to actions other than specific endonuclease detection of base damage. The issue of cell cycle-dependence was investigated, however, as in the case of EcoRl studies, there appeared to be no discrimination in terms of damage extent with respect to cell cycle position. For this reason, statistical analysis was not performed.  Table 3.12: Qualitative Comparison of Different Lysis Solutions E.A. type Hctcrog. LvsisTimc Lysis # | Enzyme | [EDTAIbf No/Yes 1 mM lOmL 0.1/1 1 hour 5 1 mM No/Yes lOmL 0.1/1 20 mins 5 0.4 No 1 hour 4 5mM lOmL 50mM lOmL 5,10,50 No/Yes/Yes 1 hour 4 5mM Agarose 50 No 1 hour 4 4 lOmM Direct 3 No 1 hour No 10 1 mM Direct 50 20/30 min 60mM Direct 50 Yes lhour 9 4 5,20 No 1 hour 1,5,10,30,100 Agarose 1 mM Direct 5 Yes 1 hour 9 5 Yes 1 hour 1, 10, 20, 75 Direct 9 E.A. refers to enzyme application described below in Table 3.13.  Table 3.13: Enzyme Application Techniques Name lOmL  Technique Total immersion in lOmL buffer plus enzyme Direct Direct application of 100, 200 or 300|iL enzyme+buffer Agarose Enzyme in agarose made with buffer  Advantage -Uniform diffusion -no drying of gel -reproducible -uses little enzyme -little contamination from storage buffer -little enzyme used -little contamination -reproducible vols.  Disadvantage -expense -enzyme adheres to plastic -storage buffer damage -uneven diffusion -excess handling required -hard to control repr.volume -additional layer reqd. -more enzyme required -second layer falls off  Results 61  Table 3.14: Base Damage Detected With Variable Concentrations of Na EDTA in Reaction Buffer 2  Dose (Gy) 0  [Enzyme] 0  irn r\i (m\l) 1 r  0.1 1.0  7  5  10 30 75 1  0  10 30 75 1  0.1 1.0 10  0  5  1 10 30 75 1 10 30 75  DNA Content 7.2 ± 1.4 8.0± 2.0 7.8 ± 2 . 2 7.2 + 2.0 7.8 ± 2 . 4 6.4± 2.2 5.9 ± 2.2 6.6 ± 1.7 6.5± 1.3 6.4 ± 1.5 5.8 ± 1.7 6.0± 2.2 5.7 ± 1.7 5.9 ± 1.7 6.5 ± 1.3 6.0 ± 1.6 6.7 ± 2.2 5.9 ± 1.8 6.2± 1,8 5.3 ± 1 . 8 5.5 ± 1.8 6.1 ± 1.5 5.6 ± 1.7 5.3 ± 1.4 5.5 ± 2 . 0 5.1± 1.7 4.9 ± 1.4 5.1 ± 2 . 1 5.7 ± 1.4  MTM 16± 1 0 2.6 ± 1.3 1.5± 1.3 5.1 ± 2 . 0 1.8 ± 1.4 9.4± 5.3 10.3 ± 7.1 2.0 ± 1.6 2.1 ± 1.3 1.4 ± 1.2 12.7 ± 5 . 3 18.3 ± 8.0 7.6 ± 4.2 4.4 ± 3.2 1.6 ± 1.2 10.5±2.1 11.4 ± 2.1 11.4 ± 1.9 13.2± 2.8 15.6 ± 3 . 5 20.0 ± 4.1 11.6 ± 2.0 11.4 ± 2.0 10.6 ± 2.1 9.4 ± 1.6 15.9 ± 3.0 17.3 ± 3.9 13.6 ± 2.1 9.0 ± 2.0  /i in Tail  c  I7.8±').7 29.8± 11.8 17.7 ±11.4 42.8± 12.9 21.1 ±10.2 59.8 ± 19.5 51.6 ±22.6 21.4± 11.4 22.9 ± 11.4 20.9 ± 10.4 69.6 ± 18.1 77.9 ± 2 1 . 1 51.6 ± 19.4 34.5 ± 16.8 22.8± 10.8 68.0 ± 8.4 59.3 ± 7 . 5 72.7±7.6 62.7 ± 8.6 82.0± 8.6 80.4 ± 8.5 64.6 ± 7.6 64.6 ± 7 . 5 62.8± 8.9 60.4 ± 7.8 82.3 ± 7 . 9 80.5 ± 19.6 73.4 ± 7 . 2 58.5± 9.4  60 110 83 110 156 94 156 70 70 64 112 100 120 200 101 110 161 150 279 88 250 70 99 101 62 100 100 175 104  Lysis solution used was 1%TX-100/1.2M NaCl/2mM N a E D T A Values shown are means and standard deviations for one or two experiments. Standard deviation was obtained from histograms generated by comet software. 2  Results 62  0 35 30 CD  E o  ^  'c6 hcCO CD  25 20 15 10 5 0 100 75  <>  50 25 0  0  4  6  8  10  Dose (Gy) Figure 3.10: Base Damage Detected as a Function of M. Luteus Crude Extract Concentration for Lysis Solution 1% Tx-100/2mM Na2EDTA/1.2M NaCl. [EDTA] in Buffer is 1mM.  Results 63  Figure 3.11: Effect of EDTA Concentration in Reaction Buffer Using 0 and 5U/uL on V79 Cells Lysed in 1% Tx-100/2mM Na2EDTA/1.2M NaCl Prior to Incubation.  Results 64 Table 3.15: Base Damage Detected With a Concentration of Na EDTA in Reaction Buffer of 60mM 2  Dose (CJy) 0  | Enzyme]  DNA Content  MTM  % in Tail  #  0  7.6 ± 1.8 6.4 ± 1.5 6.4 ± 2.0 5.6 ±1.6 7.1±2.3 6.3 ± 1.6 5.8 ± 1.9 7.1 ± 1.8 5.7 ± 1.9 6.1 ± 1.5 5.2 ± 1.8 6.0 ± 1.8 4.9 ± 1.4 5.8 ± 2 . 1 4.2 ± 1.8 5.0±1.8 5.8 ± 1.7 4.1 ± 1.8  1.2 ± 1.2 1.4 ± 1.2 4.2± 5.2 10.6 ± 4 . 6 3.4±3.5 2.3 ± 1.9 14.6 ± 4 . 9 3.5 ± 2 . 0 16.1 ± 5 . 0 5.7 ± 2 . 1 17.5 ± 3 . 9 8.4 ± 3.0 11.3 ± 2 . 7 10.9 + 2.6 24.3 ± 4 . 1 14.7 ± 4 . 5 13.6±2.4 24.7± 4.4  17 2 ± 10 5 16.5 ± 11.8 33.3 ± 19.8 51.5 ± 14.7 29.4± 17.2 23.5 ± 15.8 62.5 ± 12.8 26.8 ± 12.2 66.9 ± 13.1 37.3 ± 10.7 68.8 ± 9 . 3 54.9 ± 12.1 57.3±9.7 60.5± 9.2 86.8 ± 5 . 5 74.9 ± 11.9 63.2 ± 7 . 2 85.3± 7.1  100 200 200 200 210 110 109 111 110 110 114 110 112 125 110 284 114 158  50  1 2 4 8  50** 0 50 0 50 0 50 0 50  10  50** 0 50  Lysis solution used was 1%TX-100/1.2M NaCy2mM N a E D T A Values shown are means and standard deviations for one or two (where two values are shown) experiments. Standard deviation was obtained from histograms generated by comet software. ** Enzyme supplied by Dr. Tim Jorgensen. 2  Results 65  10 |—  r  0  2  4  6  8  10  Dose (Gy)  Figure 3.12: Base Damage Detected Using 50uL of M. Luteus Crude Extract with 60mM EDTA in Reaction Buffer. V79 Cells are Lysed Under 1 %Tx-100/2mM Na2EDTA/1.2 M NaCl Conditions. ** Crude extract supplied by Tim Jorgensen.  Results 66 Table 3.16: Base Damage Detected With Variable Concentrations of Na EDTA in Reaction Buffer. 2  Dose (Gy) 0  0.4 2 4  | EDTA |b (mM) 5(3) 10 5 10 10  2  0 3 0  10 10 5  4  0.4 3 0  10 5 10 5  1  10  |En/.ymc] 0  0.4 3 0 0.4 2 3 4 10  10 5 10 5(3) 10 5 10 10 10 5  DNA Content 5.8 ± 1.4 5.1± 1.2 5.7 ± 1.6 5.4 ± 1.4 6.2 ± 1.5 4.2 ± 1.1 4.6 ± 1.2 4.5 ± 1.6 5.0± 1.4 6.5 ± 1.9 5.2 ± 1.2 5.7 ± 1.6 4.7 ± 1.4 5.4 ± 1.4 5.4 ± 1.5 5.0 ± 1.2 6.2 ± 1.8 5.4 ± 1.4 4.8 ± 1.1 3.8 ± 1.3 5.8 ± 1.6 3.4+ 1.4 4.1 ± 1.2 5.1 ± 1.3 3.4 ± 1.3  MTM  ?, in Tail  4.5 ± 0.3 3.8 ± 2.0 12.1 ± 3 . 5 9.7 ± 2.6 13.9 ± 3.3 11.4 ± 2 . 2 4.4 ± 2.2 13.3± 6.3 5.8 ± 2 . 4 6.0 ± 2 . 6 5.8 ± 2 . 8 14.4 ± 4 . 1 13.7 ± 6 . 6 10.6± 3.5 8.7 ± 2 . 3 6.3 ± 1.7 17.6 ± 4 . 5 16.9 ± 5 . 3 14.6 ± 1.5 10.0 ± 1.8 21.2 ± 3 . 6 17.0 ± 3.4 22.1 ± 7 . 2 21.1 + 2.6 20.7 ± 4 . 8  35.6 ± 3 . 1 37.2± 14.5 70.7 ± 10.7 67.1 ± 11.4 72.9 ± 10.6 68.7 ± 9.0 41.9 ± 13.8 58.1 ±14.6 46.0 + 12.7 45.4 ± 14.6 46.6 ± 14.5 75.3 ± 8 . 8 61.4 ± 15.9 63.3± 12.2 57.7 ± 11.4 51.3 ± 10.4 83.5 ± 6 . 3 69.8 ± 9 . 8 74.5 ± 6.5 68.7 ± 7 . 6 91.2 ±5.7 85.4 ± 5 . 8 79.7 ± 8.2 89.0± 3.3 83.0± 9.2  110 209 110 110 110 114 114 120 120 111 112 112 123 121 112 121 112 112 210 57 111 119 152  Lysis used was 1% TX-100/2.5M NaCl/lOOmM Na EDTA/10mM Tris @pH 10. Values shown are means and standard deviations for one or two experiments. Standard deviations were obtained from histograms generated by comet software. Highlighted values are included in Table 3.21. 2  Results 61  0  0  4  6  8  10  Dose (Gy) Figure 3.13: Base Damage Detected as a Function of M.Luteus Crude Extract Concentration. V79 Cells were Lysed in 1%Tx-100/100mM Na2EDTA7 10mM Tris/2.5M NaCl @pH 10. [EDTA] in Reaction Buffer was 10mM.  Results 68 Table 3.17: Effect of Na EDTA Concentration in Reaction Buffer on Enzyme 2  Dose (Gy) 0  [Enzyme] 0  2  4  [EDTA]b 5(3) 10 50 10 30 100 5 10 30 100  0 0  5 50 5 50 10 5  0  10 5  10 50 1 2  4  10  10 0  2  4  10  10 50 50 5(3) 10 50 10 30 100 10 30 100 5 50  D N A Content 5.8 ± 1.4 5.1+ 1.2 3.7 ± 0 . 9 5.4 ± 1.4 4.9 ± 1.4 6.3 ± 1.5 4.6 ± 1.2 6.2± 1.5 4.2 ± 1.1 5.4± 1.5 3.3 ± 1.0 6.3 ± 1.6 4.1 ± 1.2 3.7 ± 1.5 4.1± 1.9 4.3 ±1.2 4.4± 1.2 4.6± 1.2 5.0± 1.4 6.5 ± 1.9 5.2 ± 1.2 5.4 ± 1.4 5.4 ± 1.5 5.0 ± 1.2 5.4 ± 1.3 4.9 ± 1.4 4.8 ± 1.1 3.8 ± 1.3 4.4 ± 1.2 3.4 ± 1.4 5.0 ± 1.2 4.3± 1.8 5.1 ± 1.3 5.6 ± 1.4 6.1 ± 1.6 3.4 ± 1.3 5.2 ± 1.6  MTM 4.5 ± 0 . 3 3.8 ± 2 . 0 4.3 ± 2 . 3 9.7 ± 2.6 8.8 ± 3 . 4 2.7 ± 1.8 13.9 ± 3 . 0 13.9 ± 3 . 3 11.4 ± 2 . 2 12.3±2.9 9.0 ± 3 . 6 4.6 ± 3 . 7 1.9 ± 2 . 6 21.8 ± 10.0 8.0 ± 2.6 25.0± 2.8 13.4 + 6.0 4.4 ± 2 . 2 5.8 ± 2 . 4 6.0 ± 2 . 6 5.8 ± 2 . 8 10.6 ± 3 . 5 8.7 ± 2.3 6.3 ± 1.7 10.2 ± 2.7 11.2 ± 2 . 7 14.6± 1.5 10.0 ± 1.8 15.7 ± 2 . 6 17.0 ± 3 . 4 13.9 ± 2 . 7 9.7 ± 2 . 6 21.1 ± 2 . 6 20.4 ± 2.2 15.2 ± 3 . 1 20.7 ± 4.8 16.7 ± 3 . 3  % in Tail 35.6 ± 3 . 1 37.2 ± 14.5 37.0 ± 14.1 67.1 ± 11.4 54.6 ± 14.3 27.0 ± 14.9 75.5 ± 10.9 72.9± 10.6 68.7 ±9.0 67.3± 10.2 52.6 ± 14.1 34.9 ± 19.9 22.7 ± 15.4 75.5 ± 17.0 49.6 ± 10.5 92.1 + 3.3 59.1 ± 13.2 41.9 ± 13.8 46.0 ± 12.7 45.4 ± 14.6 46.6± 14.5 63.3± 12.2 57.7 ± 11.4 51.3± 10.4 59.8 ± 10.7 60.8 ± 11.0 74.5 ± 6.5 68.7 ± 7 . 6 78.8 ± 5 . 0 85.4 ± 5 . 8 78.0 ± 7 . 6 63.8 ± 12.3 89.0± 3.3 84.1 ± 5 . 0 67.5 ± 10.2 83.0 ± 9.2 71.4± 10.2  # 110 110 110 110 114 110 110 110 114 80 114 110 173 53 109 98 114 120 120 111 123 121 112 106 105 112 105 57 117 110 119 110 110 152 106  Lysis used was 1% TX-100/2.5M NaCl/lOOmM Na EDTA/10mM Tris @pH 10. Values shown are means and standard deviations for one or two experiments. (3) show means and standard error for three independent experiments. 2  Results 69  2 uL  4uL  10  0  U  0  i  2  i  4  1  6  1  8  Dose (Gy)  U  l_i  10  0  1  2  1  4  1  6  1  8  u  10  Dose (Gy)  Figure 3.14: Effect of EDTA Concentration in Buffer Using 2 and 4uL of M. Luteus Crude Extract. V79 Cells were Lysed in 1%Tx-1007 100mM Na2EDTA/10mM Tris/2.5 M NaCl, pH 10.  Results 70  1%Tx-100, 1.2M NaCl, 2mM EDTA Lysis OGy 10 Gy  0  20 40 60 80 100 120 0 [EDTA] in Buffer mM  20 40 60 80 100 120 [EDTA] in Buffer mM  Figure 3.15: Effect of EDTA Concentration in Reaction Buffer on Enzyme Concentration for V79 Cells Unirradiated or Irradiated with 10Gy. Using Lysis (a) 1 % Tx-100/1 OOmM Na2EDTA/1 OmM Tris/2.5M NaCl @pH10 or (b) 1% Tx-100/2mM Na2EDTA/1.2M NaCl.  Results 71 Table 3.18: Base Damage Detected With a [Na EDTA ] in Reaction Buffer of 1 mM 2  Dose (G.v) 0  I Enzyme J  DN \ Content  MTM  % in Tail  0 0.1 1.0 0 0.1  7.8 ± 0 . 5 6.6 ± 1.1 6.1 ± 1.3 7.1 ± 0 . 5 6.0±2.3 6.2±1.7 6.2 ± 2 . 1 6.0 + 2.3 6.5 ± 0 . 2 6.9 + 2.4 4.6+1.7 5.8 ± 2 . 1 5.8+1.8 5.3 ± 0 . 5 4.9 ± 0.6 5.1 ± 0 . 3  2.9 ± 0.6 3.9 ± 2.4 11.4 ± 1.1 7.0 ± 2.2 8.6 + 4.7 8.9+ 3.8 15.2 ± 6 . 9 13.1 ± 6.3 11.6 ± 4.1 14.9 ± 4.6 8.2 + 3.6 24.6 ± 9.1 15.6 + 5.1 19.8 ± 7.7 21.1 ± 4.6 27.8 ± 3.2  25 6 ± 3 3 31.5±7.9 52.6 ± 6 . 2 41.0±4.0 46.2+ 14.8 56.05 ± 14.4 60.4 ± 14.5 65.8+17.9 53.8 ± 5 . 1 63.0+11.3 53.6+13.7 75.4± 12.9 75.0+12.1 74.4 ±10.6 79.4 ± 4.84 86.9 ± 3 . 1  2  1.0 4  0 0.1 1.0  10  0 0.1 1.0  #  160 213 211 211 211 189 178 187  Lysis used was 1% TX-100/1.2M NaCl/30mM Na EDTA. Two measurements. A l l others are mean and standard error for three independent experiments. Highlighted values are included in Table 3.21. 2  Table 3.19: Endonuclease III Assay Using a Concentration of Na EDTA in Reaction Buffer of ImM. 2  Dose l«.v> 0  [Enzyme |  D \ \ Content  MTM  "< in Tail  #  0  8.2 ± 2.0 8.0 ± 2 . 0 8.4 ± 2 . 2 8.4 ± 2.2 6.9 1.9 7.0 1.7 6.1± 1.6 6.4 ± 1.8  2.4 ± 1.8 2.7 ± 2 . 1 2.2± 1.3 2.1 ± 1.6 11.4 ±2.0 11.5 ± 2 . 1 12.9± 3.2 12.5± 2.6  24.0 ± 13.7 26.2 ±14.7 27.1 ± 15.0 25.5 ± 14.1 66.9 ±10.0 66.2 ± 8 . 8 67.3 ± 9 . 9 65.5 ± 8.4  113 100 103 100 94 101 201 201  l|ig/mL 8  0 l|ig/mL  Lysis solution used was 1%TX-100/1.2M NaCl/2mM N a E D T A Values shown are means and standard deviations for two independent experiments. Standard deviations were obtained from histograms generated from comet software. 2  Results 72  Figure 3.16: Base Damage Detected as a Function of M. Luteus Crude Extract Concentration. V79 Cells were Lysed in 1% Tx-100/30mM Na2EDTA7 1.2M NaCl. [EDTA] in Reaction Buffer was 1 mM.  Results 73  Figure 3.17: Base Damage Detected as a Function of Endonuclease III Concentration. [EDTA] in Reaction Buffer was 1mM. V79 cellls were used.  Results 74 Table 3.20 Base Damage Detection by Hot Alkali Treatment Lysis 0.03MNaOH,lMNaCl 0.1% Sarc.l hour room temp  O.lMNaOH, l M N a C l 0.1% Sarc. 20mins @ 50°  Dose (Gy) 0  DNA Content 8.2±3.6  MTM  % in Tail  #  1.1 ±2.2  16.7± 12.1  107  1 2 3 4 6 0  6.6 6.0 5.2 5.0 4.1 6.1  ±2.7 ±2.5 ±2.4 ±1.9 ±2.4 ±3.2  1.7 ±1.4 3.3 ±2.1 4.5 ±2.8 6.1 ±3.6 10.7±4.6 12.4± 6.2  19.3 ±11.4 27.6 ±11.0 30.4 ±11.9 35.0 ±11.8 49.5 ±13.0 49.0 ±13.7  103 104 99 101 94 110  1 2 3 4 6  5.2 4.2 4.0 5.0 4.1  ±2.3 ±2.2 ±2.5 ±2.5 ±2.1  10.0± 5.5 12.5±4.8 17.4± 7.7 15.7+ 6.2 17.6+.7.0  46.7 ±13.0 51.4 ±12.7 56.9 ±14.3 56.6 ±13.8 65.2 ±11.4  92 98 101 95 76  Values shown are means and standard deviations for one experiment only. Standard deviations were obtained from histograms generated by comet software.  Results 75  10  0  2  4  6  Dose (Gy) Figure 3.18: Radiation Dose Response (SSB) for V79 Cells Lysed in 0.03 M N a O H , 1M NaCl, 0.1% Sarcfor 1 hour @ room temperature ( O ) or 0.1 M N a O H , 1M NaCl, 0.1% Sarc for 20 mins @ 50° C ( • ).  Results 76 Table 3.21: Summary of Ness/Nssb for All Base Damage Detection Methods Lysis Solution l'cTX-100/1.2M NaCI/2mM Na EDTA  Dose (Gy) 0  I EDTA] Buffer 1 mM  Enzyme Dose 01  Ness/Nssb  1.0 5.0 0.1 1.0 5.0  0.1  3.0±3.2 6.0±4.3 0.2±0.4 0.8±0.4 0.4±0.3 1.9+1.9 0.7±1.4 0 + 1.8 0.5±0.4 0.3±0.3 0 + 0.3 0.3±0.7  1.0 0.1 1.0 0.1 1.0 0.1 1.0 2.0  3.0±0.8 0.3±0.6 1.2±0.4 0.3±0.5 1.1+0.8 0.1±0.4 0.4±0.4 1.6+1.2  4.0 2.0 4.0 lug/pX  2.6±1.7 0.7±0.4 1.1 ±0.4 0 + 0.8 0.1±0.3  0 + 1.9  2  7 10 0  10  1% TX-100/1.2M NaCl/30mM Na EDTA/10mM Tris (3)  lOmM 30mM 75mM lOmM 30mM 75mM  0  2  2 4 10 Collins Lysis: 1%TX-100/2.5M NaCl/lOOmM Na EDTA/10mM Tris; pH 10.0  0  lOmM  2  10 Endonuclease III  0 8  1 mM  Values used are highlighted in respective tables (Tables 3.14, 3.16, 3.17 and 3.19)  Discussion 11  4.  DISCUSSION  4.1  Influence of Chromatin Structure on Radiation Response Results in Fig. 3.1 indicate that V79 cells are considerably more resistant to killing by  ionizing radiation than T K 6 cells. However, in spite of these differences, there was no evidence that these two cell lines responded differently to induction of SSBs or DSBs by ionizing radiation (Tables 3.1 and 3.2). This is perhaps not surprizing since the conventional neutral comet assay requires extensive cell lysis, removing any differences in conformation structure that may affect the ability to detect D N A DSBs. In addition, ionizing radiation effectively reaches all parts of the D N A genome, and no part is resistant to double-strand breakage. However, this same argument may not hold for damage produced in D N A by the action of the restriction enzyme, E c o R l . Instead, accessibility of chromatin to this enzyme could be important, and could differ for 2 cell lines that vary in radiation sensitivity.  This  hypothesis was not supported by results presented here. Instead, access of chromatin to E c o R l appeared similar for the two cell lines, even when chromatin structure was altered by progressively increasing concentrations  of NaCl.  There are at least three possible  explanations for this result. The first is that there exists no difference between the cell lines in terms of conformationally-related accessibility, the second involves disruption of key structures by lysis methods and the third deals with comet assay sensitivity limitations. The latter two possibilitities pose serious detection problems. The first explanation, although possible in the case of artificially-induced (ie: by various non-physiological solutions; see for instance PBS vs H20) conformation, appears unlikely due to the substrate specificity required for EcoRl binding as detailed previously.  Discussion 78 Even though the EcoRl complex has been found to change its shape in order to facilitate binding, given the numerous topological possibilities as determined by sequence specificity alone, it is highly unlikely that there exist no structural differences that are responsible for accessibility to enzymes between two unrelated lines such as human and hamster cells used here. Studies comparing filter elution rate between C H O parental and its mutant xrs-5 radiosensitive line [Bryant, 1994] have shown differences  for the lines based on  conformation, presumably a structural component to D N A DSB repair. By extension, as previously pointed out by Olive [1992], this feature, which obviously remains stable under elution conditions, may not be present after stringent lysis conditions such as employed using the comet assay. Therefore, a significant drawback of this approach is that key differences, albeit subtle, would elude analysis, for example the ability to 'cope' with an introduced break (ie: retention of topology) would be obliterated under lysis.  The  two designs aimed at  addressing this issue, using radiation in conjunction with enzyme application, showed little difference in terms of enzyme accessibility, however the difference seen for irradiated untreated controls could relate to structural properties. Perhaps the additional lysis time and subsequent incubation at 37°C was responsible for this alteration; in any case, the point needs to be repeated. Alternatively, it may be that breaks are introduced, but not detected, owing in part to tangling of partly replicated D N A molecules and also to the inability to remove certain nuclear matrix proteins, which are responsible for the anchoring of loop domains thought to be involved in repair.  This is possibly another cause for the heterogeneity observed at  moderate doses of enzymes.  Discussion 79 The third explanation involves the DSB detection sensitivity of the comet assay. As stated previously in Table 1.1, the comet assay range of sensitivity for DSB detection is approximately 125-7500 DSB/cell/Gy.  Given that the average mammalian genome is  approximately 3 x l 0 bp, this translates into a detectable fragment length range of 400kb9  2.4xl0 kb. As fragments larger than 10,000kb are unable to migrate during electrophoresis, 4  essentially lengths of this size would be retained in the 'head' of the comet. Based on a study 125  correlating damage with I  decay [Olive et al, 1993], where it was assumed that one decay  produces 1 DSB, it is estimated that the comet assay detects approximately 23 DSB/cell/Gy in asynchronous V79 cells (in good agreement with results by Iliakis et al, 1991 where he reports a range of 21-31 DSB/cell/Gy).  This sets a detectable range of 5.5 - 32.6 Gy  irradiation dose and an upper mean tail moment of 45.6. E c o R l is estimated to cleave naked mammalian D N A at a frequency of every 3000bp [Bryant, personal communication], based on a mathematical probability model designed by Bishop et al., 1983]. This translates into potentially 1 million DSB sites in a cell. If every site is cut, the fragment size would be 3kb, much too small to detect. It is highly probable though, due to a number of influences including protein binding and conformational effects, that many sites are not cleaved in the assay, even at relatively high levels of salt. Results using concentrations less than 0.6M NaCl likely reflect linker D N A digestion only. This is because nucleosomal bound D N A , due to torsional stress and the number of A T pairs which are in close contact with the core, is not recognized nor cleaved.  Given that the range of  linker lengths is 14-74 bp in invertebrate D N A [van Holde, 1988], and nucleosomal lengths are 146bp (120bp attached to core, the rest mediated by histone tail domains), the relative amount of linker D N A available for recognition is about 2.7xl0 to l x l O bp or 9% to 33.2% 8  9  Discussion 80 respectively. The amount cleaved, due to the nature of the mechanism of action of E c o R l , decreases proportionally with increased cutting; cleavage takes place at an unknown distance from the recognition site and is further inhibited by short substrate length. If, however, one assumes that the frequency remains constant, the range of DSB possible is 9xl0 -3.3xl0 , 4  5  still 10-fold higher than the detection upper limit. Further complications arise when taking into consideration the fact that the enzyme can cut more than once; thus estimation of DSB probabilities are virtually impossible, especially when choosing an enzyme as a damaging agent, given substrate requirements are specific to the enzyme [Bianchi and Bianchi, 1987]. For instance, to circumvent problems arising from using a frequent cutting enzyme, the 8 bp recognition enzyme Notl was applied in a similar manner; however, this enzyme produced so little damage that a meaningful dose response range could not be obtained (data not shown), More recently, enzymes that recognize a 7 base pair sequence (e.g., SanDl) have become commercially available, and these may prove more useful than EcoRl for probing chromatin structure. It does seem quite possible, however, that key differences are being literally 'lost' as pieces < 40kb, possibly resulting from 'clustered' damage in protein-complexed D N A , are lost during lysis or migrate out of view during electrophoresis. EcoRl probably starts cleaving at recognition sites close to the surface of the nucleus, but these pieces become extensively digested and are lost while internal sequences may not be detected within the time frame of the experiment. The change in D N A content clearly supports this view. For example in 1.2M NaCl lysis prior to exposure to enzyme, the values for untreated cells were diminished by over 50% (Table 3.9) using a dosage of 0.1U/|lL. Interestingly, D N A content for permeabilized cells does not decrease appreciably with increasing tail moment, indicative  Discussion 81 of more interference caused by proteins, leading to reduced frequency of cutting. Although tail moment decreases proportionally with D N A content at high doses, no clear difference in D N A content loss rate could be seen between the lines, using D N A content as an endpoint. Heterogeneity of results, aside from obvious conformational variations in individual cells, could be due in part to patterns of diffusion of R E through agarose, however it is also likely to be due in part to heterogeneity in permeabilization of cells. A n extensive review of methods used to introduce restriction enzymes into cells is given by Bryant [1988] in which he compares the effectiveness and consequences of using inactivated Sendai viruses, osmotic shock and electroporation amongst many other methods. The consensus amongst researchers appears to be that a novel technique, which does not introduce a significant level of damage or artifacts, needs to be developed. Clearly, employing other REs, especially less frequent cutters, could provide more information on chromatin structural differences. Less frequent cutters would most likely be able to provide more insight into both quantities and spacing of nucleosomes in addition to rendering information within the detection limits of the comet assay. Nucleosomal-bound D N A could further be studied using MNase in conjunction with DNase.  One of the  drawbacks of using E c o R l in conjunction with the comet assay is that little can be inferred about the role of strength or number of nuclear matrix attachment sites, which could be particularly informative in terms of repair potential [Schwartz, 1992; Olive, 1992a]. Since REs in general show no preference for supercoiled over relaxed D N A , relaxation resulting from previous nicks would be inconsequential from the standpoint of accelerated damage caused by increased accessibility of neighbouring loop domains in radiosensitive cells [Roti Roti et al., 1993]. In addition, in order to assess D N A structure at this level, more than 3 M  Discussion 82 NaCl must be used so that the enzyme can penetrate the layers of protein, rendering so many EcoRl sites accessible that key differences near the matrix would be obscured. In any event, the null result between differences could argue, for these two particular cell lines, for an alternative explanation of radiosensitivity such as nuclear matrix involvement.  On the  positive side, the fact that conditions were developed that allowed equal EcoRl damage to two such diverse cell lines could prove very useful in studies to examine in vitro repair of D N A damage in individual cells using cellular extracts or repair complexes. These results show that all cells within a population, and both cell types, were equally susceptible to EcoRl and should therefore be equally accessible to D N A repair enzymes. As a damaging agent, the choice of EcoRl seems appropriate to induce double-strand lesions, which may be useful in assays designed to isolate this type of damage from other influences. The curve shown in Fig. 3.8 is virtually identical to the X-ray damage profile obtained by Olive and Banath [1995], who compared the response of four human tumor lines in various stages of protein depletion (Fig. 3.9) Their data shows increases in DSB lesions in H I depleted vs histone containing chromatin of 1-2 and a 4-5-fold increase after removal of H2A and H2B.  Results using EcoRl produced a 1.4 and 1.5- fold increase for H I depleted  vs Hl-containing chromatin and a 9.0 and 5.4- fold increase for H1,H2A,H2B depleted vs condensed for V79 and TK6 cells respectively. Both sets of data found decreased damage for histone-depleted structures compared to results shown by Elia and others in Table 4.1 for different reasons; the initial point used in the Olive study was 0.1M NaCl, which is far greater than the concentration used to 'condense' chromatin into its most compact form. In both cases, due to the sensitivity limit of the assay and the fact that neutral controls have a tail moment on average between 1 and 2, it is unlikely that a 39.5-fold increase could be scored.  Discussion 83  In conclusion, numerous studies indicate that chromatin conformation influences the response of mammalian cells to ionizing radiation. The general approach was to determine whether radiation sensitivity could be predicted on the basis of a simple measure of chromatin accessibility. However, probing chromatin structure using the restriction enzyme, E c o R l , did not provide discriminatation between a radioresistant cell line and a radiosensitive cell line. In future experiments, other restriction enzymes (i.e., less frequent cutters) may prove more useful.  Table 4.1: Summary of Studies of the Effect of DNA Conformation on Author Elia and Bradley, 1992; Xue et al, 1994; Olive andBanath, 1995 Elia and Bradley, 1992; Xue et al, 1994 Xue etal, 1994 Warters and Lyons, 1990, 1992  4.2  Structure comparison  Fold increase in DSB Lesions  HI depleted: H I containing  2-3 fold  H I , H2A,H2B: condensed Histone depletedxondensed Expanded:Condensed  10-16 fold 39.5 fold 4-5 fold  Detection of Base Damage Using the Comet Assay Efforts to develop a method to detect radiation-induced D N A base damage were  largely unsuccessful.  A major problem was that a significant amount of base damage was  present or introduced in unirradiated controls, possibly due to damage occurring during different lysis stages. The results shown for alkali-labile lesions produced under normal vs high temperature/NaOH (Table 3.20 and Fig. 3.18) circumstances are consistent in terms of slopes obtained with enzyme data; treatment with enzyme shifts the radiation dose-response curve upwards parallel to the untreated curve. This effect is independent of lysis solution used (Figs. 3.10, 3.13, 3.14, 3.16) or level of E D T A added to the buffer (Figs. 3.11, 3.12, 3.15). What is puzzling is that more damage in irradiated controls treated with enzyme is not being detected in addition to these breaks; no significant loss of D N A , which would indicate  Discussion 84 severe fragmentation, was observed even after treatment with high enzyme concentrations. Further, the upper limit of mean tail moment (-45) was not reached for all cells.  It is  possible that not enough enzyme was used to saturate all base damage sites, however, in order to increase the protein concentration significantly, further purification would be necessary [Jorgensen et al, 1988]. An alternative explanation is that this crude extract, which houses at least 5 different endonuclease activities [Schon-Bopp et al., 1977], is only cleaving at certain sites within D N A . This 'native' endonuclease has been found by Schon-Bopp et al. [1977] to cleave in this manner in unirradiated D N A . It may be that the other endonucleases, specifically the Tendonuclease, which cleaves at altered base structures, are simply not recognizing the substrate for numerous reasons, including the involvement of residual proteins not removed by these lysis conditions. Summarized in Table 4.2 are reported findings using both M.luteus extracts and endonuclease IJJ for the detection of base damage. Significantly, the literature reveals that most of the work has been done using U V irradiation, which is known to lead to the production of a high level of thymine glycols, however, even these reports are finding a 10fold decrease from most H P L C estimates. Possibly, as Collins believes, the results for H P L C techniques are artificially high because oxidation of guanine occurs during isolation, storage or hydrolysis or D N A [Collins, 1996]. Alternatively, there could be a detection problem arising from the insensitivity of most methods used in conjunction with this approach: alkaline filter elution, sucrose sedimentation and unwinding. Table 4.3 shows sensitivity ranges in terms of detectable single strand breaks for numerous methods. As previously outlined, these methods, including  Discussion 85 the comet assay, have drawbacks; an important one is damage by exposure to alkali. A detailed analysis of the literature further reveals interesting discrepancies. Skov, et al. [1979, 1984] reported a Ness/Nssb for aerobic conditions of 0.9 (1979) for U V light and using alkaline sucrose gradient sedimentation only a moderate enhancement in base damage was seen for X-rays at 40 Gy.  No control (unirradiated) data were reported for either study and  the scatter in the data is large, which they admit, yields only an approximate result. It is likely, however given the low-end of sensitivity for this technique, that this was unable to detect background breaks. Studies by van Loon, et al [1993] using alkaline filter elution to characterize X-ray induced base damage, may also fall into this category. For both dose response and repair curves, the difference seen in treated vs untreated controls was on average l S S B / 1 0 D . This y  translates into a M T M of approximately 8. In addition, his results show a great deal of scatter, indicative perhaps of heterogeneity influences, but more likely a result of taking very few measurements. Due to insensitivity of the assay (lOObreaks/cell), the lowest data point taken is for lOGy. More convincing results are presented by Bryant [1978], who used the more sensitive alkaline unwinding method in conjunction with two methods of enzyme delivery; osmotic shock and a non-ionic detergent permeabilization (Brij-58) for UV-treated cells. As this is one of the few studies which retains chromatin structure, it is interesting to note that his results show not only reduced background, but also more effective detection of base damage. In contrast,  Fohe and Dikomey [1994], who also use unwinding, report a high level of  control damage. It is possible that even short duration of lysis, such as used in this technique, is detrimental to detection.  Discussion 86 The use of the comet assay to detect base damage produced by H 0 has been reported 2  2  by Collins et al. [1993] using endonuclease UJ. Their technique involved lysis in very high salt/Na EDTA as described previously, and it is not clear whether rinsing of salt was 2  adequate. As reported previously [Fairbairn et al., 1995], inadequate rinsing of NaCl can result in comets which appear much less damaged due to high salt gradients during electrophoresis. This would account for some of the heterogeneity present in his data; however, as the collection process is done by visual means and binned into only 5 comet classes, it is difficult to evaluate the data. Another consideration that has not yet been taken into account is the effect of the agarose itself. It is possible that agarose, which contains some toxic contaminants, may produce A P sites which might not be detectable as strand breaks (ie: not alkali-labile). Bryant's method of introducing enzyme directly into permeabilized cells seems to be effective in terms of both accessibility and low background damage. A technique of this kind, possibly involving the use of agarose-encapsulated beads or more a gentle means of agarose mixing following permeabilization, may prove useful in reducing damage. In conclusion, the application of endonucleases to detect D N A base damage using the comet assay should be feasible, based on previous results in the literature.  However,  problems with background damage, alkali-induced damage, cellular heterogeneity, and defining optimum conditions for enzyme activity are not insignificant. required to reach this goal.  Further work is  Discussion 87  Table 4.2 Summary of Studies of Base Damage Detection Using Endonucleases Ref.  Hiizynie T Agent  Collins, et EndoIB al, 1993, 1995 van Loon, m.luteus et aZ.,1991, fraction I 1993  1 H2O2  2  Cell Type  Ness/Nssb  Untreated control  Treated control  1-leLu  n/a  50/10 D  50/10'-D  110/10 D 400/10 D  670/10 D 2800/10 D  Lymphocytes U.V. Human 0.51±0.14 wbc  EndoUI  12  12  Skov, K . A m.luteus 1984 fraction I 1979  m.luteus fraction I  2 3  Hamster 0.5 ±0.1 n/a bone marrow U.V. PM2 0.37+0.03 <0.1 D N A <0.1DNA bacteriophage modifications/ modifications /10 bp 10 bp 4  1.48±0.13 4  U.V.  3  U.V.  5 X-rays  5  UV  n/a  80% in dsf  90% in dsf  CHO  ERS:55.1 SB:133.9 0.41 n/a  <0.1 breaks/DNA molecule n/a  <0.1 breaks/DNA molecule n/a  CH2B2  0.9  n/a  n/a  0.16 ESS 0.13 SSB n/a  n/a  n/a  1.7±0.4  0.55 in dsf  0.9 in dsf  MRC-5 human pUC18 plasmid  X-rays Wilkins,R. J 1973 Fohe,C. and Dikomey, E.,1994  I2  00  4  m.luteus fraction I Bryant, m.luteus P.E.,1978 fraction I Jorgensen m. luteus etal, 1988 fraction I  12  n/a 7-rays  Epe et al, 1993  12  m. luteus fraction I  5  U.V.  m.luteus fraction I  4 X-rays  Human fibroblast CHO  T= Technique. 1. Alkaline Comet Assay 2. Alkaline Elution 3. HPLC/relaxation 4. Alkaline unwinding 5. Alkaline sucrose gradient sedimentation.  Discussion 88  Table 4.3 Methods Used to Detect DNA SSBs in Mammalian Cells Technique  Zonal rotor gradient sedimentation Alkaline elution Alkaline unwinding  Sensitivity Approx. (breaks/cell) Time Required 50-1000  8 hours  100-5000 50-50,000  24 hours 4 hours  Alkaline D N A precipitation Nucleoid sedimentation Halo Assay  500-25,000  2 hours  100-20,000  2 hours  200-15,000  2 hours  Alkaline Comet Assay  50-15,000  6 hours  Nick translation  250-20,000  2 hours  Expense  r.ffoit  References  Moderate  High  Ueno, A , M . et al., 1979  Moderate High Kohn,1991 Low Moderate Ahnstrom and Erixonl973; Rydberg, 1980 Low Low Olive, 1988 Low  Moderate C o o k & Brazell,1975 Moderate Moderate Roti Roti, and Wright, 1987; Taylor et al, 1991 Moderate Moderate Fairbairn et al, 1993; 01ive,P.L. etal, 1990 Low Moderate Fertil,B. et al, 1984; Nose and Okamoto,1983; Snyder and Matheson,1985  Estimates of range of sensitivity are based on response to ionizing radiation, with 1 Gy producing 100 SSB/diploid cell Effort reflects technical proficiency required and amount of time required per sample. Reprinted with permission from Molecular Approaches for Detecting D N A Damage, Olive, P.L., 1997.  Summary and Conclusions 89  5.  SUMMARY  5.1  EcoRl-Induced Damage Using the Comet Assay The technique of using restriction enzymes as an adjunct to comet analysis has been  optimized in terms of method of application and lysis conditions.  Results using salt  extraction (Table 3.10, Figs. 3.9 and 3.10) have shown that restriction enzymes are useful as damaging agents, effectively mimicking double-strand damage by ionizing radiation (Fig. 3.10). In addition, in conjunction with dose-response results, chromatin conformational response to the ionic strength of the medium is consistent with results in the literature. This can be seen from the initial dip of the curve (Fig. 3.9) and the response to successive removal of protein and the consistency of results which are in accord with literature values (Table 4.1). Chromatin structure can be effectively assessed at many levels of organization. The most informative data would be found using a frequent cutter or MNase on cells permeabilized with non-ionic detergent and also an infrequent cutter on cells treated with more than 0.6M NaCl.  To gain information about nuclear matrix proteins, it might be  possible to use an infrequent cutter on cells treated with 3M NaCl. Clearly the use of E c o R l and elevated levels of salt was not informative in terms of T M , however further digestion studies could be performed using D N A content as an endpoint. Some feature of TK6 cells yet unidentified could be responsible for the observed loss of D N A seen in Fig.3.7. Under permeabilizing conditions, heterogeneity was minimal, reproducibility was high and linearity of response was consistent; further work substituting another enzyme could prove useful. Heterogeneity of the results (ie: damage differences in individual cells) still poses a big problem with respect to detection.  It is unclear whether the cause is non-uniform  Summary and Conclusions 90 penetration of cells, enzyme concentration or kinetics. The responses using three methods of application did not differ appreciably and it is difficult to determine whether or not enzyme saturation level is responsible; cells heavily damaged with a high concentration of enzyme are detected at the high end of comet analysis sensitivity so any further damage would most likely be lost. The most feasible method of application is direct pipetting onto agarose and covering with a coverslip. To make this more economical, gels can be made smaller by partitioning the slide with a trace of hydrophobic silicone lubricant. The conclusion from these studies is that EcoRl is not a useful probe for detecting differences in chromatin structure that relate to intrinsic radiosensitivity. As a frequent cutter, it may fail to detect large scale differences in chromatin packaging, or results may simply be an indication that there are no significant differences in chromatin accessibility between cell types that differ in radiation response.  Before abandoning this hypothesis, it  would be important to test less frequent cutters.  5.2  Base Damage Detection Using the Comet Assay A method for base damage detection was developed, which could be easily  incorporated into the comet assay for the purpose of assessing oxygen enhancement ratios. The  ideal lysis environment in terms of minimal background damage, cost and  reproducibility is 1%TX-100, 2mM N a E D T A and 1.2M NaCl and the amount of N a E D T A 2  2  in the reaction buffer which inactivates nonspecific activity but does not interfere with specific enzyme properties is lOmM. Inefficient rinsing of salt leads to artificially low damage detection and in addition to retardation effects.  Summary and Conclusions 91 Clearly the use of crude M. leuteus extract was insufficient for the purposes of detection using X-rays, however, there is enough evidence in the literature to suggest that it may be useful in detection of UV-irradiated base damage.  A second purification step,  outlined by Jorgensen et al., 1988, which would be only initially cumbersome to set up and of minimal expense, is a necessary refinement for this approach. It is likely that all of the damage caused by the extract results from aspecific activity.  Either the specific y-  endonuclease is not recognizing the substrate base damage or (AP) sites are being created by the assay. Both of these point to agarose involvement; this is supported by the fact that Bryant's method, which involves lysis stages similar to the ones used in this study, yields successful results. It is possible that either agarose has some contaminants which produce A P sites or that it inhibits enzymatic cleavage. Mild permeabilization of cells in suspension with a non-ionizing detergent and enzyme prior to agarose mixing would eliminate both effects. Alternatively, agarose beads may be used. The conclusion from these studies is that detection of base damage using the comet assay, while feasible, suffers from a number of technical problems.  Further studies are  required to understand the nature of the background damage and to define the optimum conditions for detection of base damage in comets using purified repair enzymes.  References 92  6.  REFERENCES  Ahnstrom, G. Techniques to measure D N A single-strand breakss in cells: a review [Review]. International Journal of Radiation Biology 54: 695-707, 1988. Arents, G. and Moudrianakis, E.N. Topography of the histone octomer surface: repeating structural motifs utilized in the docking of nucleosomal D N A . Proc. Natl. Acad. 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Linker D N A accessiblility in chromatin fibers of different conformations: a reevaluation. Proc. Natl. Acad. Sci. USA 91: 5277-5280, 1994.  APPENDIX Critical values (Fcrit and tcrit) for A N O V A and t Test Statistical Analysis  dfw  ANOVA Fcrit <0.1  Fcrit <0.5  df  18.51 10.13 7.71 6.61 5.99 5.59  98.49 34.12 21.20 16.26 13.74 12.25  2 3 4 5 6 .7  tTest tcrit < 0.05 tcrit < 0.01 tcrit < 0.001  (dfb=l)  2 3 4 5 6 7  4.606 3.182 2.776 2.571 2.447 2.365  9.925 5.841 4.604 4.032 3.707 3.499  31.598 12.924 8.610 6.869 5.959 5.408  Values obtained from statistical tables in Kranzler and Moursund, 1995.  


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