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A DNA-binding protein from Hela cells which binds preferentially to DNA damaged with ultraviolet light… Tsang, Siu Sing 1981

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A DNA-BINDING PROTEIN FROM HELA CELLS WHICH BINDS PREFERENTIALLY TO DNA DAMAGED WITH ULTRAVIOLET LIGHT OR N-ACETOXY-N-ACETYL-2-AMINOFLUORENE by SIU SING TSANG B.Sc, M c G i l l U n i v e r s i t y , 1976 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MEDICAL GENETICS We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1981 SIU SING TSANG, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Medical Genetics The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 2 5 t h M ay> 1 9 8 1 • n F - f i 10 /7Q) Abstract A DNA-binding protein, PHI, was partially purified from extracts of Hela c e l l s by high-speed centrifugation, and chromatography on DEAE-cellulose, phosphocellulose and UV-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the UV-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. PHI binds preferentially to supercoiled PM2 DNA treated with ultraviolet light (UV-DNA) or N-acetoxy-N-acetyl-2-aminofluorene (AAAF-DNA) as compared to native supercoiled PM2 DNA. The binding i s noncooperative. A filter-binding assay u t i l i z i n g GF/C glass fibre f i l t e r s was used to detect PHI during the purification steps. Characterisation of PIII-DNA complex by glycerol gradient centrifugation indicates that the retention of the complex by the f i l t e r s does not involve DNA precipitation, aggregration, or a conformational change of the DNA which results in a detectable change in the sedimentation coefficient of the DNA. The binding of PHI to DNA i s reversible. PHI is a protein as indicated by i t s sensitivity to proteinase K. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation i s 2.0-2.5 S corresponding to a molecular weight of about 20-25,000 i f the protein i s spherical. The binding between UV- or AAAF-DNA and PHI i s optimal at around 100-200 mM NaCl and i s relatively independent of temperature and pH. MgCl 2 and MnCl 2 at concentrations between 1 mM and 7 mM do not markedly affect the binding but i t i s inhibited by sucrose, ATP and caffeine. i i i Competition experiments indicate that PHI i s a single protein which binds to AAAF-induced and UV-induced DNA binding sites with equal a f f i n i t y . PHI also binds preferentially to supercoiled PM2 DNA treated with N-methyl-N'-nitro-nitrosoguanidine but has l i t t l e or no preferential binding activity for methyl methanesulphonate-treated or depurinated PM2 DNA. It also possesses some binding activity to unit length, single-stranded PM2 DNA. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates for PHI, indicating a requirement of DNA supercoiling for the binding activity of PHI. The possible nature of the DNA-binding sites for PHI i s discussed. The biological significance of PHI remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease ac t i v i t i e s . The binding of PHI does not alter the susceptibility of UV-irradiated supercoiled PM2 DNA to the single-stranded endonuclease of Neurospora crassa. A DNA-binding protein similar to PHI was found to be present i n extracts of a normal human fibroblast c e l l line and two xeroderma pigmentosum fibroblast c e l l lines (XP-cell lines). The concentration of this protein i n the extracts of these c e l l lines was comparable to that of PHI i n Hela c e l l s . The two XP-cell lines were XP5EG and XP2NE. They belong to the A and D complementation groups of xeroderma pigmentosum, respectively. The c e l l line XP5EG appeared to be deficient i n another DNA-binding protein, which eluted from the phosphocellulose column with 180-250 mM potassium phosphate. The dissociation equilibrium constant for the binding reaction of P H I to the UV- or AAAF-induced binding s i t e s on DNA i s estimated to be 7 x 10 ^ M. The a s s o c i a t i o n r a t e constant and the d i s s o c i a t i o n rate constant are 4 x 10^ M *sec * and 3 x 10 ^sec \ r e s p e c t i v e l y . There are at l e a s t 10"* molecules of P H I per Hela c e l l . V Table of Contents Page Abstract 11 Table of Content v List of Tables v i i i L i s t of Figures x i Acknowledgement x i i Abbreviations x i i i Introduction 1 Materials and Methods 7 1. Tissue culture 7 (a) Cell lines 7 (b) Culture media 7 (c) Solutions for harvesting cells 7 (d) Cell growth 8 2. Preparation of ^-labeled PM2 DNA 9 3. Preparation of modified DNA 9 4. DNA-binding assay 11 5. Precycling and preparation of column resins 11 (a) DEAE-cellulose and phosphocellulose 11 (b) UV-irradiated DNA-cellulose 12 6. Purification of the DNA-binding protein, PHI 13 (a) Crude extract 13 (b) DEAE-cellulose chromatography 13 (c) Phosphocellulose chromatography 14 (d) UV-DNA-cellulose chromatography 14 VI 7. Analysis of DNA-binding proteins from human fibroblasts 15 8. Glycerol gradient sedimentation of PHI 16 9. Glycerol gradient sedimentation of PIII-DNA complex 16 10. Sucrose gradient sedimentation of DNA 17 11. Enzyme assays 17 12. Protein determination 19 13. Phosphate determination for the column fractions 19 14. Sci n t i l l a t i o n f l u i d 19 15. Miscellaneous 19 Results 21 1. Purification of the DNA-binding protein, PHI 21 2. Properties of the DNA-binding assay 26 3. Formation of PIII-DNA complex as a function of the amount of DNA damage and the concentration of PHI 33 4. Substrate specificity 42 5. Other properties of PHI 51 6. Glycerol gradient sedimentation analysis of PHI 66 7. Characterisation of the PIII-DNA complex 69 8. Catalytic activity 77 9. DNA-binding proteins i n normal human and XP-fibroblasts 80 10. Estimation of the equilibrium constant of the binding reaction and the concentration of PHI 85 Discussion 89 1. Advantages of using glass fibre f i l t e r s in the f i l t e r -binding assay 89 v i i 2. Mechanism of retention of PIII-DNA complex by the GF/C f i l t e r s 90 3. Comparison of PHI with other UV- or AAAF-DNA-binding proteins from human cells 91 4. Biological significance of PHI 92 5. Nature of the binding site for PHI 94 Bibliography 98 v i i i L i s t of Tables Table Page I. P u r i f i c a t i o n of P H I from Hela c e l l s 22 I I . Retention of PIII-DNA complex by d i f f e r e n t types of Whatman glass m i c r o f i b r e f i l t e r s 32 I I I . E f f i c i e n c y of retention^of PIII-DNA complex by the GF/C f i l t e r s 37 IV. E f f e c t of DNA conformation on the DNA-binding a c t i v i t y of P H I 52 V. Estimation of DNA damage on the various DNA substrates 53 VI. Substrate s p e c i f i c i t y of P H I 54 VII. S e n s i t i v i t y of P H I to proteinase K and RNase A treatment 55 VIII. E f f e c t of temperature on the DNA-binding a c t i v i t y of P H I 59 IX. Freeze-thaw s t a b i l i t y of P H I 60 X. E f f e c t of sucrose and g l y c e r o l on the DNA-binding a c t i v i t y of P H I 65 XI. Assay f o r DNA-endonuclease a c t i v i t y of P H I 78 XII. Assays for UV-DNA endonuclease and glycosylase a c t i v i t i e s of P H I under various conditions 79 XIII. Preparation of extracts used f o r the analyses of the DNA-binding proteins from human f i b r o b l a s t s 82 XIV. Summary of the analyses of a UV-DNA-binding protein i n human f i b r o b l a s t extracts 86 lx L i s t of Figures F i g . Page 1. Chromatography of DNA-binding proteins on phosphocellulose 23. 2. UV-DNA c e l l u l o s e chromatography of the phosphocellulose f r a c t i o n of P H I .. 25. 3. E f f e c t of NaCl concentration i n the assay mixture on the DNA-binding a c t i v i t y of P H I 27. A. Time course of DNA-binding by P H I 28. 5. Retention of PIII-DNA complex on the f i l t e r s as a function of the NaCl concentration of the d i l u t i o n buffer 29. 6. E f f e c t of f i l t r a t i o n speed on the retention of PIII-DNA complex 31. 7. DNA-binding of P H I as a function of UV-dose and AAAF-dose 34. 8. DNA-binding as a function of the amount of P H I 35. 9. S p e c i f i c binding of UV-DNA and AAAF-DNA with various amounts of P H I 39. 10. DNA-binding as a function of the amount of P H I at a low low concentration of DNA substrates 40. 11. S p e c i f i c binding of UV-DNA with various amounts of P H I at a low concentration of DNA substrates 41. 12. DNA binding curve at low concentrations of P H I 43. 13. Binding of P H I to DNA i r r a d i a t e d with high UV-doses .... 44. 14. AAAF-DNA binding a c t i v i t y of P H I i n the presence of competitor DNA 45. X 15. A reciprocal plot of the data of the competition experiment depicted in Fig. 14 47 16. Sucrose gradient sedimentation of Msp I-treated DNA 48 17. Effect of MgCl 2 and MnCl 2 on the binding activity of PHI 57 18. DNA binding activity of PHI: pH dependence 58 19. Heat sensitivity of PHI 61 20. Effect of ATP on the DNA-binding activity of PHI 63 21. Effect of caffeine on the binding activity of PHI 64 22. Sedimentation velocity analyses of PHI in the presence of 0.15 M NaCl 67 23. Sedimentation velocity analyses of PHI in the presence of 0.5 M NaCl 68 24. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol, and 0 mM NaCl 70 25. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol, and 50 mM NaCl 71 26. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol, and 150 mM NaCl 72 27. Sedimentation of PIII-u-DNA complex in 10-30% glycerol, and 0 mM NaCl 74 28. Sedimentation of PIII-UV-DNA complex formed in the presence presence of ATP and MgCl 2 75 29. Reversibility of the binding of PHI to UV-DNA 76 x i 30. Effect of PHI on the susceptibility of DNA to the single-stranded specific endonuclease from Neurospora cvassa 81 31. Phosphocellulose chromatography of DNA-binding proteins from human fibroblast extracts 83 x i i Acknowledgement I thank Dr. U. Kuhnlein f o r supervising my research. I t has been stimulating and rewarding to work with him. I am also g r a t e f u l to Dr. H. F. S t i c h f or h i s constant support and Dr. R. M i l l e r , Dr. G. Tener, and Dr. S. Wood f o r devoting t h e i r time to be on my supervisory committee. Mrs. G. Wood a s s i s t e d i n some of the tiss u e culture work and Mrs. J . Koropatnick prepared the 3H-labeled PM2 DNA. My wife, Francoise, deserves s p e c i a l thanks f o r her patience, encouragement and help. Studentship awards received from the National Cancer I n s t i t u t e of Canada during the period of 1978-81 are g r a t e f u l l y acknowledged. Abbreviations AAAF N-acetoxy-N-acetyl-2-aminofluorene AAAF-DNA PM2 DNA treated with AAAF ADP adenosine diphosphate ATP adenosine triphosphate ATPase adenosine triphosphatase BSA bovine serum albumin CLL chronic lymphocytic leukemia DEAE-cellulose O-(diethylaminoethyl) cellulose DMSO dimethylsulfoxide DNA deoxyribonucleic acid DTT dithiothreitol EDTA disodium ethylene diaminetetraacetate MMS methylmethanesulphonate MMS-DNA PM2 DNA treated with MMS MNNG N-methyl-N'-nitro-nitrosoguanidine MNNG-DNA FM2 DNA treated with MNNG MNUA N-methyl-N-nitrosourea rpm revolution per min S sedimentation coefficient Tris tris-(hydroxymethyl)-aminomethane u-DNA untreated native PM2 DNA UV ultraviolet light UV-DNA PM2 DNA UV-irradiated UV-endonuclease endonuclease which cleaves DNA adjacent to pyrimidine dimers XP xeroderma pigmentosum 1 Introduction The s t r u c t u r a l and fu n c t i o n a l i n t e g r i t y of the DNA genome i n a c e l l i s sometimes a l t e r e d by DNA damage which can a r i s e either spontaneously (1) or by the act i o n of chemical and p h y s i c a l agents (2-4). I f the l e s i o n s are not corrected by DNA r e p a i r processes, normal DNA metabolism and gene regulation w i l l be affected. Several types of DNA re p a i r processes have been proposed and reviewed (5, 6). Information concerning these processes has l a r g e l y been obtained from studies with procaryotes. However, human c e l l s probably also r e p a i r DNA damage by s i m i l a r processes. Several r e p a i r - d e f i c i e n t human genetic diseases have been i d e n t i f i e d (7, 8). In some of these diseases, the patients are cancer prone (9). Among them, xeroderma pigmentosum i s probably the best characterised (10, 11). Patients with xeroderma pigmentosum (XP) are very s e n s i t i v e to sunlight and a l l of them have the tendency to develop skin tumors. C e l l l i n e s have been established from the skin f i b r o b l a s t s of XP patients. Except f o r a group of XP c e l l s c a l l e d XP va r i a n t , the f i b r o b l a s t s of these X P ' c e l l l i n e s have been shown to be defective i n the e x c i s i o n r e p a i r of UV-induced thymidine dimers. In normal c e l l s , e x c i s i o n r e p a i r of the pyrimidine dimer i s believed to be i n i t i a t e d by an i n c i s i o n on the DNA i n the v i c i n i t y of a dimer. The i n c i s i o n i s made eit h e r by a s p e c i f i c endonuclease a c t i v i t y (UV-endonuclease a c t i v i t y ) or v i a a combination of a glycosylase a c t i v i t y and an apyrimidinic endonuclease a c t i v i t y (12). The DNA damage and adjacent nucleotides are then removed by an exonuclease. The gap thus created i s then f i l l e d with a DNA polymerase a c t i v i t y and f i n a l l y the 2 repair patch i s joined to the remaining DNA by a ligase (5, 6). The excision repair deficiency in the XP cells seems to l i e in the incision step of the process (10, 11, 13). Defects of XP.cells in other.DNA repair processes have also been reported (14-17). Cell hybridization studies indicate that the excision repair defect in XP c e l l lines f a l l s into at least seven complementation groups (10). This finding suggests that the incision step of the excision repair pathway for pyrimidine dimers is a complex process. Furthermore, those XP cells which repair the pyrimidine dimer deficiently are also defective in the repair of bulky DNA lesions caused by other "UV-like" DNA damaging agents such as AAAF and bromobenzanthracene (8, 11). These XP cells however can repair proficiently DNA lesions incurred by other damaging agents such as MMS and X-rays (8, 11). The DNA damaging agents of the latter group each e l i c i t s a short repair patch size of about 3-4 nucleotides in a c e l l ; whereas with the former group of agents, the repair patch size may be as long as 120 nucleotides (6, 18). It i s possible that the excision repair of DNA lesions introduced by UV and by the "UV-like" DNA damaging agents may share the same repair enzymes or some regulatory proteins. Other studies have also suggested the existence of regulatory molecules in chromatin which might determine the removal of pyrimidine dimer6 from DNA. It was found that extracts of XP cells from the complementation groups A and D and the XP variant were capable of excising thymidine dimers from purified UV-irradiated DNA. In contrast, extracts from ce l l s of the XP group A and the XP variant did not excise dimers from their endogenous chromatins under conditions where extracts 3 of normal cells and XP group D cells did (19, 20). However, these results contradict the repair capacity of intact c e l l s , where the XP -variant cells but not the XP group D cells exhibit normal excision repair (21). Nevertheless, i t was suggested that the XP cells are not defective in the UV-endonuclease activity, which must act before the dimers are excised. Rather, there may be factors which affect the recognition of DNA damage in chromatin by the UV-endonuclease, and some XP c e l l s may have a deficiency in one or more of these factors (19, 20). An added complexity for DNA repair in human cells i s imposed by the chromatin structure. Basically the chromatin structure is composed of repeating units of nucleosome core particles with the DNA wrapped around octamers of histones. These core particles are connected by the linker-DNA (22). DNA lesions in the nucleosome core are less accessible to DNA repair enzymes than the linker DNA (21, 23-27). For human cel l s irradiated with ultraviolet light, i t has been calculated that the probability of repair synthesis per unit length of DNA in the linker regions i s 15-fold greater than that in the core particles, while there i s no predominance of induction of pyrimidine dimers in the linker regions (27). It has also been shown that both the UV-endonucleases of Micr-ococens tuteus and phage T4 have limited access to the dimer sites i n permeable irradiated human cells (26). Additional sites became accessible when the cells were exposed to a high concentration of NaCl which presumably disrupts the chromatin structure. Thus, the incision step in the human excision repair process i s carried out by an endonuclease and by factors which control the accessibility 4 of DNA damage to the putative repair endonuclease. A protein which appears to influence the rate of DNA incision by a UV-endonuclease has been identified in human lymphocytes from patients with chronic lymphocytic leukemia (CLL) (28). This protein was purified by DNA-cellulose chromatography. It eluted from a UV-irradiated calf thymus DNA-cellulose column with 1 M NaCl and from a single-stranded DNA-cellulose column with 2 M MaCl. It had a molecular weight of 24,000. This protein can enhance the melting or unwinding of poly[d(A-T)] and UV-irradiated calf thymus DNA but not native calf thymus DNA. Interestingly, the rate of cleavage of UV-irradiated supercoiled 4>X-174 vj^A by the UV-endonuclease activity of Micrococcus luteus was enhanced by this unwinding protein. Using an immunochemical procedure, this protein was not detected in lymphocyte extracts from normal individuals. The presence of this protein might explain the higher DNA repair capability of CLL cells compared with normal cells (28). We and others have so far failed to purify a pyrimidine dimer specific endonuclease activity from human ce l l s . These failures may be due to the small quantity or the l a b i l i t y of the endonuclease activity in crude extracts of human cel l s (19, 29). It i s possible that the UV-endonuclease activity i s a complex of several protein molecules, which dissociates upon chromatography leading to a loss of endonuclease activity. An analogous situation exists for the UV-endonuclease activity coded by the uvrA, B and C genes of Escherichia coli. Of the three UV-endonuclease a c t i v i t i e s purified from procaryotes, the one coded 5 by the uvrA, B and C genes in Escherichia coli i s probably the best model for the UV-endonuclease activity i n human c e l l s . Unlike the UV-endonucleases of Micrococcus luteus and phage T4 which are specific for pyrimidine dimers, the UV-endonuclease of Escherichia coli recognizes bulky DNA adducts. Such adducts are repaired less e f f i c i e n t l y in XP c e l l s . Mutations in the uvrA, B or C genes render Escherichia coli sensitive to both UV light and to agents which can produce bulky DNA adducts (6, 30, 31). It has been shown that each of the uvrA, B or C gene products does not have an appreciable endonuclease activity. They however can complement each other to yield an ATP-dependent endonuclease activity specific for UV-irradiated DNA (32). The uvrA protein apparently has a molecular weight of 100,000. It binds to UV-irradiated superhelical DNA and to a lesser extent to unirradiated superhelical DNA (33). Recently, a dimer specific endonuclease activity has been isolated from calf thymus (34). It is labile and i s probably associated with a high molecular weight complex. It i s l i k e l y that proteins which bind strongly to DNA damaged by UV or other agents have a role i n DNA repair. Thus, one approach to isolate proteins which function i n DNA repair i s to assay for their binding a b i l i t i e s to damaged DNA. The simplest assay involves incubation of the protein with DNA i n a reaction mixture and subsequent f i l t r a t i o n of the mixture through a nitrocellulose f i l t e r . The protein-DNA complex i n the reaction mixture i s retained by the f i l t e r . Such filter-binding assays have been shown to be useful in the purifications of several proteins which are involved or may be involved in DNA repair. These proteins included the UV-endonuclease activity from Micrococcus luteus (35), the T4 endonuclease V (36), 6 the UVYA protein (33), an ATP-independent UV-endonuclease from Escherichia c o l i (37) , a DNA-binding protein which can insert purines into apurinic sites (38, 39) and the apurinic endonuclease activity from human fibroblasts (38). The filter-binding assays have also allowed the purification of two human placental DNA-binding proteins. Their biological functions remain to be determined. One of them binds to UV-irradiated DNA but recognizes DNA lesions other than pyrimidine dimers (AO). It also binds to DNA treated with nitrous acid or sodium bi s u l f i t e ( A l ) . The other protein binds efficently to DNA treated with either AAAF, MMS or MNUA but has no a f f i n i t y towards UV-irradiated DNA (A2). Recently, glass fibre f i l t e r s have also been used in the f i l t e r -binding assays of three DNA-binding proteins. The three proteins are the DNA-terminal protein of adenovirus (A3, AA), a protein from Hela cells which binds tightly to cellular DNA with an average spacing of about 50,000 base-pairs (AA) and the poly(ADP-ribose) polymerase from bovine thymus which binds to DNA containing single- or double-stranded breaks (A5). The last protein might have a role i n DNA repair (A6, A7). In the hope that we may be able to isolate an activity which i s involved i n the incision step of excision repair of bulky DNA adducts, we have attempted to purify DNA-binding proteins from Hela cells which bind preferentially to UV-DNA and AAAF-DNA. In this thesis, the partial purification and characterisation of such a DNA-binding protein i s reported. We have developed a filter-binding assay using GF/C glass fibre f i l t e r s for the assay of this DNA-binding protein. In addition, fibroblasts from a normal human c e l l line and c e l l lines of XP group A and XP group D were screened for the presence of this DNA-binding protein. 7 Materials and Methods 1 1. Tissue culture (a) Cell lines Hela c e l l s were purchased from Flow Laboratories, Inc., Rockville, Maryland. Cell line 207 was a g i f t from Dr. S. Wood, Department of Medical Genetics, University of British Columbia. It i s derived from a skin biopsy from a 32 year old normal male Caucasian. XP c e l l lines were obtained from the Human Genetic Mutant Cell Repository, Institute of Medical Research, Camden, New Jersey. Cell line XP5EG belongs to the A complementation group of xeroderma pigmentosum and was derived from a skin biopsy of a 23 years old white female. Cell line XP2NE belongs to the D complementation group of xeroderma pigmentosum and was derived from a skin biopsy of a 4 year old white male of Egyptian background born of consanguineous parents. (b) Culture media Minimal essential Eagle's medium (Gibco) vas supplemented routinely with 10% fe t a l calf serum (Gibco) and the following antibiotics: p e n i c i l l i n (100 ug/ml), streptomycin sulphate (30 ug/ml), kanamycin (100 ug/ml) and fungizone (2.5 ug/ml). The antibiotics were purchased from Gibco. The medium was adjusted to pH 7.0-7.5 with sodium bicarbonate. The culture medium was sterilized by f i l t r a t i o n through a GSWP Millipore membrane f i l t e r with a pore size of 0.2 ym. (c) Solution for harvesting c e l l s Trypsin-EDTA solution was prepared with 8.0 gm of NaCl, 0.2 gm of KH2P04, 0.2 gm of KCl, 1.15 gm of Na2HP04, 0.2 gm of EDTA and 0.5 gm of trypsin (Trypsin 1:250, Difco) and 1 l i t e r of double d i s t i l l e d water. The solution was sterilized by f i l t r a t i o n through 8 a GSWP Millipore f i l t e r . 1 l i t e r of phosphate buffered saline (PBS, pH 7.1) contained 8 gm of NaCl, 0.2 gm of KC1, 1.15 gm of Na2HP0^ and 0.2 gm of KB^PO^ and was st e r i l i z e d by autoclaving. (d) Cell growth 2 Hela c e l l s were maintained i n 75-cm tissue culture flasks (Falcon Plastics) with 15 ml of culture media. Four flasks of confluent c e l l s were pooled and used to inoculate ten rol l e r bottles (Bellco Biology 2 Glassware). Each r o l l e r bottle has a surface area of 840 cm . 100-150 ml of culture medium was used i n each bottle. The r o l l e r bottles were incubated at 37°C and rotated at a speed of 0.1-0.2 rpm. After 4-6 days, the c e l l s were harvested. The c e l l culture medium was decanted, and the c e l l s were washed briefly with 10 ml of the trypsin-EDTA solution. The Hela c e l l s then were detached from the surface of the bottles by incubation with another 10 ml of the trypsin-EDTA solution for 5-10 min at room temperature. During this period the ro l l e r bottles were rotated at a speed of 3 rpm. The ce l l s were pelleted by centrifugation at 200-400 g for 6 min. The pellet was washed three times with 10 ml of PBS by repeated resuspension and pelleting. The f i n a l c e l l pellets were stored i n liquid nitrogen. Ten bottles normally 9 gave 0.5-1.0 x 10 c e l l s . Human fibroblasts were grown i n plastic tissue culture flasks 2 (Nunc Company) with a surface area of 174 cm . The volume of the culture medium was about 30 ml. Incubation was at 37°C i n a humidified incubator In an atmosphere of 5% C0 2 and 95% a i r . Confluent c e l l s were sp l i t 1:3. Cells were harvested near confluency i n lots of 24 flasks 8 with a yield of 0.5-1.0 x 10 c e l l s . The c e l l s culture medium was 9 decanted, and the c e l l s were washed for 2-5 min with 5 ml of the trypsin-EDTA solution. Cells were detached from the tissue culture flask by incubating with another 5 ml of the trypsin-EDTA solution for 5-10 min at 37°C. They were washed and pelleted with PBS as described for Hela c e l l s . 3 2. Preparation of H-labeled PM2 DNA The PM2 DNA was prepared as described previously (48) except that the Pseudomonas Bal-31 bacteria were infected at a c e l l density of 3-5 g x 10 cells/ml with a multiplicity of infection of 10 phage per bacterium 3 instead of 2-4 phage per bacterium, and that 2 mCi/liter of methyl- H-thymidine (specific activity, 25 Ci/mmol, Amersham) was used to label the PM2 DNA. The higher multiplicity of infection was found to increase the yield of the PM2 phage. The PM2 DNA had a specific activity of 17-22,000 cpm/yg of DNA. 3 Unlabeled DNA was prepared in the same way as the H-labeled DNA except that no radioactive thymidine was added. 3. Preparation of modified DNA UV-irradiation of DNA was carried out at a DNA nucleotide concentration of 0.5 mM in 10 mM Tris-HCl, pH 7.5, using a petri dish on ice and a 60 watt GE G15T8 germicidal lamp. The incident dose was measured with a Blak-ray ultraviolet meter (Ultraviolet Products, Inc.). 2 The standard dose used was 1,200 J/m . AAAF-DNA was prepared by incubating PM2 DNA at a DNA nucleotide concentration of 0.5 mM at 37°C for 1 h with various concentrations of AAAF (a g i f t from Dr. J. Scribner, Fred Hutchinson Cancer Research Centre, Seattle) i n 10 mM Tris-HCl, pH 7.5, and 10% DMSO. The AAAF-DNA used In the standard DNA-binding assay was prepared with 0.01 mg/ml AAAF. The DNA then was d i l u t e d to a nucleotide concentration of 0.2 mM and dialys e d overnight against two changes of 500 ml of 10 mM Tr i s - H C l , pH 7.5. Depurination of PM2 DNA was ca r r i e d out by heating DNA at a nucleotide concentration of 0.5 mM at 70°C f o r 15 min i n 10 mM T r i s , 0.1 M NaCl and 0.01 M sodium c i t r a t e at a pH of 5.0 (adjusted with HCl). The treatment created about 1.5 apurinic sites/PM2 DNA molecule as determined by the nicking assay of Kuhnlein et a l . (49). MNNG-DNA and MMS-DNA were prepared by incubating PM2 DNA at a nucleotide concentration of 1 mM i n 10 mM Tr i s - H C l , pH 7.5, with various concentrations of MNNG or MMS for 30 min at 37°C. Supercoiled c i r c u l a r PM2 DNA was converted to a l i n e a r form by incubating PM2 DNA at a nucleotide concentration of 0.1 mM for 3 h with 32 units/ml of r e s t r i c t i o n endonuclease Msp I (New England Biolabs) i n 10 mM Tr i s - H C l , pH 7.5, 10 mM MgC± 2, 6 mM KC1 and 100 pg/ml of acetylated BSA. In experiments where single-stranded DNA was used, the l i n e a r PM2 DNA was extracted f i r s t with an equal volume of chloroform-octanol (9:1) and then dialysed overnight against two changes of 400 ml of 10 mM T r i s - H C l , pH 7.5. The l i n e a r PM2 DNA was denatured immediately before use by a 10-min incubation i n a b o i l i n g water bath. Nicked PM2 DNA was prepared by tre a t i n g the supercoiled c i r c u l a r PM2 DNA with bovine pancreatic DNase I (Sigma). Native PM2 DNA at a nucleotide concentration of 0.1 mM was incubated with 6.5 ug/ml of DNase I at 37°C fo r 1 h i n a reaction mixture containing 10 mM Tr i s - H C l , pH 7.5, 10 mM NaCl and 100 ug/ml of acetylated BSA. A f t e r t h i s treatment v i r t u a l l y a l l the DNA molecules were nicked. 4. DNA-binding assay The standard DNA-binding assay mixture contained 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 139 fmol of 3H-labeled PM2 DNA molecules (14,000-18,000 cpm), 175 mM NaCl and an aliquot of protein i n a total volume of 300 y l in a borosilicate test tube. The mixture was incubated for 10 min on ice. The assay mixture then was diluted with 1.7 ml of ice cold 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl (buffer G), and filtered immediately through a GF/C f i l t e r at a flow rate of 10-30 ml/min. The f i l t r a t i o n speed was controlled by a Manostat Varistaltic pump. The reaction tube was rinsed once with 1.7 ml of buffer G, and the resulting solution was f i l t e r e d . The f i l t e r funnel (Millipore) and the f i l t e r then were washed with another 1.7 ml of buffer G. F i l t e r s were dried under a heat lamp and the radioactivity was determined by liquid s c i n t i l l a t i o n counting. A unit of DNA-binding activity i s defined as the amount of protein which retains 1 fmol of PM2 DNA on the f i l t e r under the standard conditions. 5. Precycling and preparation of column resins (a) DEAE-cellulose and phosphocellulose The two kinds of resins were precycled i n the same way. Routinely, 100 gm of resin was suspended i n 2 l i t e r s of d i s t i l l e d water in a beaker. The resin was allowed to settle for about 1 h and the supernatant containing fine particles was decanted. The procedure was repeated three times, and the resin was resuspended i n 1 l i t e r of 0.5 M NaOH for 20 min. The suspension was f i l t e r e d through a Whatman No. 1 f i l t e r paper. The resin then was washed with d i s t i l l e d water u n t i l the f i l t r a t e had a neutral pH. It was stirred with 2 l i t e r s of 10 mM potassium phosphate, pH 7.5, and l e f t at 4°C overnight. The suspension 12 was f i l t e r e d and washed with d i s t i l l e d water. F i n a l l y , the r e s i n was resuspended i n 10 mM potassium phosphate, pH 7.5, and stored at 4°C. (b) UV-irradiated DNA-cellulose C e l l u l o s e (Cellex 410, Bio-Rad) was precycled as described by Alb e r t s and Herrick (50). 100 gm of c e l l u l o s e was suspended i n 1 l i t e r of ethanol and incubated at 80°C for an hour. The c e l l u l o s e was allowed to s e t t l e , and the ethanol was poured o f f . The procedure was repeated three times. The c e l l u l o s e then was successively washed by suspending and f i l t e r i n g at room temperature with 500 ml each of 0.1 M NaOH, 1 mM EDTA and 10 mM HCl. A f t e r washing with H 20 u n t i l the pH of the e f f l u e n t was neu t r a l , the c e l l u l o s e was l y o p h i l i z e d and stored at room temperature. A s o l u t i o n containing 2 mg/ml of c a l f thymus DNA i n 10 mM Tris-HCl pH 7.4, and 1 mM EDTA (buffer X) was prepared. 40 ml of the DNA s o l u t i o n i n a polypropylene beaker with a diameter of 5 cm was UV-irradiated for 35 min with a G15T germicidal lamp. The incident UV-dose 2 was 12 j/m /s. The DNA so l u t i o n was mixed vigorously with a magnetic s t i r r e r during the i r r a d i a t i o n . 20 g of the l y o p h i l i z e d c e l l u l o s e was added to the UV-irradiated DNA s o l u t i o n . The lumpy mixture was spread out on a glass d i s h with a glass rod. The d i s h was covered with gauze and a i r dried at 37°C overnight. Afterwards the DNA-cellulose was ground to a powder and l y o p h i l i z e d overnight to complete the drying procedure. The dry DNA-cellulose was resuspended i n 20 ml of 95% ethanol and UV-irradiated at a dose rate of 10 J/m2/s for 20 min. The DNA-c e l l u l o s e was a i r - d r i e d again at 37°C overnight. The DNA-cellulose then was resuspended i n 1 l i t e r of buffer X and l e f t at 4°C f o r a day. I t was washed twice by resuspension and f i l t r a t i o n with 2 l i t e r s of buffer X to remove free DNA. F i n a l l y , the DNA-cellulose was resuspended i n 100 ml of buffer X plus 0.15 M NaCl and stored as a frozen s l u r r y at -20°C. 6. P u r i f i c a t i o n of the DNA-binding p r o t e i n , P H I A l l operations were at 4°C. The columns were made from B-D p l a s t i c syringes. D i a l y s i s was ca r r i e d out with Spectrapor I d i a l y s i s tubing with a molecular weight cut off of 6,000-8,000. The column f r a c t i o n s were c o l l e c t e d i n polypropylene or polyethylene tubes. (a) Crude extract 9 About 2 x 10 Hela c e l l s were used f o r the p u r i f i c a t i o n of P H I . Hela c e l l s were thawed and suspended i n 35 ml of 50 mM T r i s - H C l , pH 7.5, 1 mM EDTA and 1 mM DTT (buffer A). The Hela c e l l s were disrupted by sonication with s i x 20-sec pulses using a Biosonik I I I sonicator (Bronwill S c i e n t i f i c ) . The sonication was performed at an i n t e n s i t y s e t t i n g of 30 using a 4 mm probe. The sonicate was centrifuged for 50 min at 50,000 rpm i n a Beckman 50 T i r o t o r . The supernatant was centrifuged once more under i d e n t i c a l conditions to insure complete removal of a l l sedimentable m a t e r i a l . The f i n a l supernatant (high speed supernatant f r a c t i o n ) was retained f o r further p u r i f i c a t i o n . (b) DEAE-cellulose chromatography 2 A column (3.8 cm x 5.3 cm) with 20 ml of Whatman DE-22 DEAE c e l l u l o s e was prepared and e q u i l i b r a t e d with 50 mM T r i s - H C l , pH 7.5, 1 mM EDTA, 1 mM DTT, 10% g l y c e r o l and 0.4 N NaCl (buffer B). The high speed supernatant f r a c t i o n was brought to the same buffer content and loaded onto the column at a flow rate of 0.25-0.5 ml/min. The column then.was washed with buffer B at the same flow r a t e . Fractions of 10 ml were c o l l e c t e d . A t o t a l of 10-12 f r a c t i o n s were c o l l e c t e d and assayed f o r DNA-binding a c t i v i t y . The f r a c t i o n s with a c t i v i t y were 14 pooled and dialysed overnight against two changes of 1 l i t e r of 10 mM potassium phosphate, pH 7.4, 10% gl y c e r o l , 1 mM DTT, 1 mM EDTA (buffer C). The whitish precipitate (appearing after 1-2 h of d i a l y s i s ) was removed by centrifugation at 15,000 rpm for 15 min i n a Beckman 50 T i rotor. The supernatant (DEAE-fraction), about 60 ml, was retained for further p u r i f i c a t i o n . (c) Phosphocellulose chromatography 2 The DEAE-fraction was applied to a 45-ml column (5.5 cm x 8 cm) of Whatman P - l l phosphocellulose previously equilibrated with buffer C. The column then was washed with 30 ml of buffer C. Fractions of 10 ml were collected. The column subsequently was washed with 50 ml of 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 1 mM DTT and 10% glycerol (buffer D). Afterwards the column was eluted with a 400-ml l i n e a r gradient from 50 mM to 500 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA, 1 mM DTT and 10% glycerol. Fractions of 6 ml were collected. The flow rate was 0.3-0.5 ml/min. The column fractions were made 40% i n glycerol and stored at -20°C. Fractions containing DNA-binding a c t i v i t y eluting between 325-425 mM potassium phosphate were pooled (phosphocellulose fraction) and subjected to further chromatography. (d) UV-DNA cell u l o s e chromatography 2 A column (0.6 cm x 5 cm) with 3 ml of UV-irradiated DNA-cellulose was equilibrated with 10 mM Tris-HCl, pH 7.5, 10% gly c e r o l , 1 mM EDTA and 1 mM DTT (buffer E). A 13-ml aliquot of the phosphocellulose f r a c t i o n was made 100 ug/ml i n B-lactoglobulin and dialysed against 1 l i t e r of buffer E for 3-4 h. The dialysed extract was applied to the DNA-cel l u l o s e column at a flow rate of about 0.25-0.5 ml/min. The column was washed with 3 ml of buffer E containing 100 yg/ml of B-lactoglobulin (buffer F) followed successively by 10 ml each of buffer F containing 0.15 M NaCl, 0.5 M NaCl, 1 M NaCl or 2 M NaCl. Fractions were stored i n 55% glycerol and 100 ug/ml of B-lactoglobulin at -20°C. 7. Analysis of DNA-binding proteins from human fibroblasts The purification procedures were modified from those for Hela c e l l s . Briefly, about 5-6 x 10^ human fibroblasts were used in each analysis. They were disrupted by sonication i n 4 ml of buffer A as described for Hela c e l l s . The sonicate was centrifuged at 50,000 rpm for 50 min in a Beckman 50 T i rotor. The supernatant was passed 2 through a column (0.64 cm x 3.3 cm) of 2 ml of DEAE-cellulose. The DEAE-cellulose column then was washed with buffer B at a flow rate of 0.17 ml/min. Fractions of 1 ml were collected. DNA-binding activity was detected i n the f i r s t 7-8 fractions. These were combined and dialysed overnight i n a Spectrapor I dialysis tubing against two changes of 500 ml of buffer C. The dialysed extract was centrifuged at 15,000 rpm for 15 min i n a Beckman 50 T i rotor to remove a precipitate formed during the dialysis. The supernatant (DEAE-fraction) was 2 chromatographed on a 3.5-ml phosphocellulose column (1.1 cm x 3.2 cm). The column was washed with about 3 ml of buffer C and 3-5 ml of buffer D. Fractions of 1 ml were collected. The column then was eluted with a 30-ml linear gradient from 50 mM to 500 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA, 1 mM DTT and 10% glycerol at a flow rate of about 0.17 ml/min. Fractions of 0.5 ml were collected and B-lactoglobulin was added to a f i n a l concentration of 200 yg/ml. The column fractions were assayed immediately for DNA-binding activity. 16 8. Glycerol gradient sedimentation of PHI 0.2 ml of a diluted aliquot of PHI was layered on a 4.8 ml, 10-30% linear glycerol gradient in a polyallomer centrifuge tube. The glycerol gradient was buffered with 10 mM Tris-HCl, pH 7.5, and contained 1 mM EDTA, 1 mM DTT, 0.15 M NaCl and 100 ug/ml bacitracin. Where indicated, the gradient solution contained 0.5 M NaCl instead of 0.15 M NaCl. Gradients were centrifuged at 49,000 rpm and 4°C for 27 h in a Beckman SW 50.1 rotor. Fractions were collected in polypropylene tubes from the bottom of the gradient. BSA (4.25 S), egg white ovalbumin (3.5 S), a-chymotrypsin (2.5 S), whale skeletal muscle myoglobin (2.0 S) and cytochrome C (1.7 S) from Sigma Chemical Company were used as marker proteins. The molecular weights of these proteins are 64,000, 45,000, 24,300, 18,000 and 12,400 respectively. The sedimentation profile of these marker proteins was monitored by measuring the absorbance at 280 um and i n the case of cytochrome C, also at 410 um. A symmetrical peak of absorbance was obtained for each protein. 9. Glycerol gradient sedimentation of PIII-DNA complex Unless otherwise stated, the DNA-binding reaction was performed under the standard conditions. A 200-pl aliquot of the assay mixture was layered on a 4.8 ml, 10-30% linear glycerol gradient containing 10 mM Tris-HCl, pH 7.5, 0.1 mM DTT, 1 mM EDTA and 100 ug/ml 3-lactoglobulin. In some experiments, the gradient solutions contained 50 mM or 150 mM NaCl. Gradients were centrifuged at 49,000 rpm and 4°C in a Beckman SW 50.1 rotor for 2 or 3 h. Fractions were collected from the bottom into polypropylene tubes. A 50-yl aliquot from each fraction was assayed for radioactivity. The remaining portions of the peak fractions of each gradients were fi l t e r e d through GF/C f i l t e r s as described for the standard DNA-binding assay. The amount of PIII-DNA complex was determined by the amount of radioactive PM2 DNA retained on the f i l t e r s . 10. Sucrose gradient sedimentation of DNA A 200-yl aliquot of the DNA solution was layered on a 4.8 ml 5-20% linear sucrose gradient containing 50 mM Tris-HCl, pH 7.5, and 0.25 M NaCl. Centrifugation was for 3 h at 50,000 rpm and 4°C in a Beckman SW 50.1 rotor. Fractions were collected from the bottom. A 50-ul aliquot from each fraction was assayed for radioactivity. 11. Enzyme assays Endonuclease activity was assayed by measuring the conversion of supercoiled PM2 DNA to nicked ci r c l e s . The standard assay for DNA nicking was according to the method of Kuhnlein et a l . (16, 49). 50 y l of the reaction mixture was diluted with 150 y l of 0.01% of sodium dodecyl sulphate, 2.5 mM EDTA (adjusted to pH 7.0 with HC1). 200 y l of 0.3 M K2HP04-KOH, pH 12.5, then was added. After 2 min at room temperature, the solution was neutralized with 100 y l of 1 M K^PO^-HCl, pH 4.0. This treatment denatured nicked PM2 DNA, but not covalently elosed circular PM2 DNA. 200 y l of 5 M NaCl and 5 ml of 50 mM Tris-HCl, pH 8.0, and 1 M NaCl then were added successively. The solution was f i l t e r e d through a nitrocellulose membrane f i l t e r paper (Schleicher and Schuell type BA 85, 0.45 ym pore size) which selectively retained denatured DNA. The f i l t e r was washed with 5 ml of 0.3 M NaCl, 0.03 M sodium citrate and dried. The amount of DNA retained on the f i l t e r was. determined by measuring the radioactivity on the f i l t e r by liquid s c i n t i l l a t i o n counting. The average number of nicks per DNA molecule (u>) was calculated from the equation 10 = -ln(l-X) (16), where X i s the fraction of nicked PM2 DNA molecules. Glycosylase activity was measured by determining the number of a l k a l i - l a b i l e apurinic/apyrimidinic sites introduced into the DNA. The assay for a l k a l i - l a b i l e sites was similar to the assay for DNA nicking except that the 2-min alkali-treatment was replaced by a 1 h incubation with 200 pl of 0.3 M ^HPO^-KOH, pH 12.5, and 50 mM L-lysine at 37°C. This procedure hydrolyses apurinic/apyrimidinic sites (16, 40). Exonuclease activity was measured by determining the amount of radioactive DNA rendered soluble in 6% trichloroacetic acid. For the assay of ATPase activity, aliquots of PHI were incubated in a reaction mixture containing 139 fmol of PM2 DNA molecules, 1 mM ATP, 2.5 mM MgCl 2 > 10 mM Tris-HCl, pH 7.5, and 0.8 uCi/ml of 2, 8-3H-ATP (25 Ci/mmol, New England Nuclear). After incubation for 1 h at 37°C, the conversion of ATP to ADP was monitored by the method of Romberg et a l . (51). In this method aliquots of the reaction mixtures are analysed by thin layer chromatography on strips (0.6 cm x 6 cm) of polyethyleneimine cellulose (Brinkmann) with a solution of 1 M formic acid and 0.5 M L i C l at room temperature. The chromatography procedure separates ATP from ADP: ATP remains near the origin, and ADP migrates to the middle of the strip. The strips were cut into two portions to determine the amount of radioactive ATP and ADP in the reaction mixture. 12. Protein determination Protein concentrations were determined by the method of Lowry et a l . (52) or the method of Bradford (53). For the latter assay, the dye was purchased from Bio-Rad Laboratories. BSA (Sigma) was used as a protein standard in both methods. 13. Phosphate determination for the column fractions The reagent solution for phosphate determination contained one volume of 10% ascorbic acid and six volumes of a solution containing 0.42% ammonium molybdate and 1 N I^SO^. For each assay, 1.4 ml of the reagent solution was mixed with 0.6 ml of the sample to be tested. After incubation for 20 min at 45°C, the mixture was cooled to room temperature and the absorbance at 660 um was determined (54). Inorganic phosphate solutions were used as standards. The absorbance was linear between 10 and 100 nmol of phosphate. 14. Sci n t i l l a t i o n f l u i d F i l t e r s were counted in toluene (BDH Chemicals) containing 4 gm/1 of 2,5-diphenyloxazole (PPO) (Amersham) and 0.1 gm/1 of 1,4-bis[2-(5-phenyloxyazolyl)]-benzene (POPOP) (Syndel Laboratory). For aqueous samples ACS s c i n t i l l a t i o n liquid (Amersham) was used. 15. Miscellaneous Proteinase K (20 mAnson units/mg) was purchased from E. Merck Biochemicals. Bovine pancreatic ribonuclease (Type IA, 76 Kunitz units/mg) and the single-stranded specific endonuclease of Neurospora erassa (535 units/mg) were purchased from Sigma. BSA was acetylated with acetic anhydride (Fisher) as described previously (49). Caffeine, MnCl 2 > T r i s (Trizma), ATP and ADP were obtained from Sigma. MgCl-2, sucrose and g l y c e r o l were purchased from F i s h e r . Unless otherwise stated, a l l pH measurements were performed at room temperature. Results 1. Purification of the DNA-binding protein, PHI Details of the purification procedures are described in Materials and Methods. The results of a typical purification are summarised in Table I. 9 A high speed supernatant fraction was prepared from 2 x 10 Hela c e l l s and was f i l t e r e d through a DEAE-cellulose column in buffer B which contained 0.4 M NaCl. The DEAE-fraction had a higher DNA-binding activity than the high speed supernatant fraction. This result might reflect a removal of cellular DNA or another inhibitor of DNA-binding activity by f i l t r a t i o n through the DEAE-cellulose. The DEAE-fraction was fractionated by phosphocellulose column chromatography. Three major peaks of DNA-binding activity were separated (Fig. 1). The activity which eluted at 325-425 mM potassium phosphate bound preferentially to UV- or AAAF-DNA as compared to the untreated DNA (u-DNA); with a ratio of 6:1. This peak of DNA-binding activity was stable for at least a year when stored at r20°C in the presence of 40% glycerol. The two other peaks of DNA-binding activity eluted in the flow-through fractions (data not shown) and with 180-250 mM potassium phosphate. They did not show any binding specificity to UV- or AAAF-DNA under our present assay conditions. Fractions 59-71 of the phosphocellulose column were pooled (phosphocellulose-fraction) and further fractionated by DNA-cellulose chromatography. The UV-irradiated DNA-cellulose column was eluted with a step gradient. The major species of DNA-binding protein, designated a r b i t r a r i l y as PHI, eluted with 1 M NaCl from the column 22 Table I. Purification of PIII from Hela c e l l s . Activity, Units of activity x 10 Protein Volume units x 10 per mg of protein Fraction mg ml UV-DNA u-DNA UV-DNA u-DNA I. High speed supernatant 350 40 540 250 1.6 0.7 II. DEAE 300 60 750 400 2.5 1.3 III. Phospho-cellulose 2.6 117 1 270 45 104 17 IV. UV-DNA-cellulose _ 2 54 3 95 14 - -Volume of the fraction in 40% glycerol. •The amount of protein was not determined. 'Volume of the fraction in 55% glycerol. 23 F r a c t i o n F i g . 1. Chromatography of DNA-binding proteins on phosphocellulose. Assays were performed with UV-DNA ( • ) , AAAF-DNA (•) or u-DNA ( O ) as the binding substrates. Phosphate concentration ( ) . (Fig. 2). This purification step separated PHI from another DNA-binding activity which eluted from the column with 0.5 M NaCl. Peak fractions of PHI were stored in 55% glycerol and 100 ug/ml 6-lactoglobulin with no loss in DNA-binding activity for at least half a year. Routinely, 100 ug/ml of 6-lactoglobulin was included in the elution buffers for the DNA-cellulose chromatography of PHI. It has been reported that carrier proteins such as lysozyme affect the elution of steroid receptors on DNA-cellulose chromatography (55). We have not observed any effect of 6-lactoglobulin on the elution of PHI from the UV-irradiated DNA-cellulose column. A similar elution profile as that shown in Fig. 2 was obtained when the chromatography was carried out with elution buffers without B-lactoglobulin. However, i f stored in the absence of 6-lactoglobulin, PHI lost at least half of i t s activity in 16 h at 4°C. Other attempts which included the storage of PHI in the absence of carrier protein at -20°C or in liquid nitrogen with and without 55% glycerol failed to stabilize PHI. BSA or acetylated BSA can be used to stabilize PHI, but we have chosen a r b i t r a r i l y 6-lactoglobulin. The purification of PHI after phosphocellulose chromatography was at least 40-fold. The ratio of AAAF- or UV-DNA-binding activity relative to u-DNA-binding activity increased from about 2:1 in the f i r s t two fractions to about 7:1 in the f i n a l fraction. This indicates an enrichment of the specific binding activity for AAAF-DNA and UV-DNA during the purification. 0.15 M 0.5 M 1.0 M 2.0 M N a C l N a C l N a C l N a C l - i * * * F r a c t i o n F i g . 2. UV-DNA c e l l u l o s e chromatography of the phosphocellulose f r a c t i o n of P H I . Assays were performed with UV-DNA ( • ) , AAAF-DNA (•) or u-DNA ( O ) as the binding substrates. 26 2. Properties of the DNA-binding assay Basically, the DNA binding assay consisted of four steps: (1) incubation of PHI with DNA; (2) dilution of the assay mixture; (3) f i l t r a t i o n of the mixture through GF/C glass fibre f i l t e r s ; (4) washing of the f i l t e r and f i l t e r funnel with f i l t r a t i o n buffer. The standard conditions for the DNA-binding assay were established by studying several parameters. F i r s t , the influence of NaCl concentration on the DNA-binding activity of PHI was investigated. Fig. 3 indicates that the binding of PHI to UV- or AAAF-DNA was optimal at 100-200 mM NaCl. Thus, PHI can bind optimally to UV-or AAAF-DNA at ionic strength near physiological conditions. A salt concentration of 175 mM NaCl was used in the standard assay mixture. The binding of PHI to DNA was very fast and an incubation of two min at 0°C was sufficient for the establishment of an equilibrium (Fig. 4). Therefore, our standard incubation condition of 10 min was more than adequate. The next condition we studied was the NaCl concentration of the dilution buffer used in the second step of the DNA-binding assay. It should be noted that the binding reaction could s t i l l occur in this second step. The result depicted in Fig. 5 indicated that the amount of UV-, AAAF- or u-DNA retained on the f i l t e r by PHI was maximal at 20-50 mM NaCl. Since we were interested primarily in the specific binding of PHI to UV-DNA and AAAF-DNA, a dilution buffer with 0.1 M NaCl was chosen to minimize the nonspecific binding of PHI to DNA and/or the retention by the f i l t e r s of complex formed via the nonspecific binding of PHI to DNA (Fig. 5). The former was more l i k e l y since the GF/C f i l t e r can retain the adenovirus terminal 27 0 1 0 0 2 0 0 3 0 0 N a C l , m M Fig. 3. Effect of NaCl concentration in the assay mixture on the DNA-binding activity of PHI. The DNA-binding assays were performed with the standard assay mixture except that the NaCl concentration was varied as indicated. After incubation at A C for 10 min, the assay mixtures were diluted with 1.7 ml of 10 mM Tris-HCl buffer (pH 7.5) containing NaCl to give a f i n a l concentration of 100 mM NaCl. The assays were then completed as described for the standard DNA-binding assay. DNA-binding activity was measured with UV-DNA (• ) , AAAF-DNA ( • ) or u-DNA ( O ) . 28 F i g . 4. Time course of DNA-binding by P H I . DNA-binding assays were performed under the standard conditions f o r various incubation times with UV-DNA ( • ) , AAAF-DNA (•) or u-DNA ( O ) as the binding substrates. 29 50 150 250 N a C l , m M F i g . 5. Retention of PIII-DNA complex by the f i l t e r s as a function of the NaCl concentration of the d i l u t i o n b u f f e r . The DNA-binding assays were performed under the standard conditions except that the NaCl concentration i n the d i l u t i o n buffer was varied as indicated. DNA-binding a c t i v i t y was assayed with UV-DNA (• ), AAAF-DNA (• ) or u-DNA ( O ) . protein complex with an equal efficiency at various NaCl concentrations from 0.15 M to A M NaCl (A3, AA). A similar phenomenon has been observed in the binding of bovine thymus poly(ADP-ribose) polymerase to nicked DNA. A salt concentration of 0.1 M NaCl also was used to inhibit the nonspecific binding of the polymerase to closed DNA (A5). We found that f i l t r a t i o n speeds faster than 30 ml/min or slower than 5 ml/min resulted in a substantial loss of the amount of PIII-DNA complex retained on the f i l t e r s (Fig. 6 ) . Thus we usually fi l t e r e d at a flow rate of 10-30 ml/min. It i s well documented that a slow flow rate results in a more reproducible and greater retention of protein-DNA complex by nitrocellulose f i l t e r s (56, 57). The reason for the loss of PIII-DNA complex with low f i l t r a t i o n speeds remains to be determined. There are five different kinds of Whatman GF grade glass fibre f i l t e r s which differ in their thicknesses and pore sizes (Table II). The pore size of each f i l t e r i s defined as the size of the particles that can be retained by the f i l t e r with an efficiency of 98% (Glass microfibre f i l t e r s , Whatman Publication 82A). Different kinds of f i l t e r s were tested for their ability to retain PIII-DNA complex. Filters with pore sizes less than 1.5 ym retained the PIII-DNA complex eff i c i e n t l y (Table II). Usually, assays were performed in conditions where 5-25% of the input UV-DNA or AAAF-DNA was retained by the f i l t e r s . In experiments where the assays were carried out in duplicate, the duplicates usually agreed to within 10%. In the absence of PHI, the amount of DNA retained on the f i l t e r s 31 F i g . 6. E f f e c t of the f i l t r a t i o n speed on the retention of P I I I -DNA complex. The binding-assays were performed with UV-DNA (•) or u-DNA ( O ) at the various f i l t r a t i o n speeds indicated. Each points i s the average of duplicate assays. The f i l t r a t i o n time was the time needed f o r a 2-ml assay mixture to pass through a f i l t e r . 32 Table I I . Retention of P I I I - D N A complex by different types of Whatman glass microfibre f i l t e r s DNA retained on f i l t e r , fmol Type Thickness, mm pore size, ym UV-DNA u-DNA GF/A 0 . 2 5 1 . 6 1 1 . 5 1 . 9 GF/B 0 . 7 1 1 . 0 1 3 . 2 4 . 0 GF/C 0 . 2 5 1 . 2 1 2 . 0 2 . 9 GF/D 0 . 6 5 2 . 7 8 . 5 2 . 8 GF/F 0 . 4 4 0 . 7 1 2 . 2 2 . 4 The DNA-binding assays were performed under the standard conditions with different types of Whatman glass fibre f i l t e r s . The data were the averages of duplicate assays. 33 (background) was l e s s than 1.5% for the various kinds of duplex PM2 DNA we have used. The background for the unit length single-stranded PM2 DNA was even lower and was 0.3-0.5% of the input DNA. Where appropriate, the backgrounds were substracted from the amount of DNA retained on the f i l t e r s i n the presence of P H I . 3. Formation of PIII-DNA complex as a function of the amount of DNA damage and the concentration of P H I An a l i q u o t of P H I which contained 23.5 units of UV-DNA-binding a c t i v i t y only retained 2.4 fmol of u-DNA on the f i l t e r . The amount of PIII-DNA complex retained on the f i l t e r increased when the DNA was treated with increasing dose of UV or AAAF ( F i g . 7). The maximum amount of PIII-DNA complex was retained when the UV-dose and the 2 AAAF-dose reached 1,200 J/m and 0.01 mg/ml, re s p e c t i v e l y . The r e s u l t 2 also suggested that DNA UV-irradiated with a dose of 1,200 J/m was equivalent to DNA treated with 0.01 mg/ml AAAF as a binding substrate for P H I . Thus, UV-DNA and AAAF-DNA were prepared accordingly and used i n experiments where the binding of P H I to both DNA substrates were compared. Under the standard conditions, the retention of DNA was l i n e a r l y dependent on the amount of P H I (Fig. 8). However, at higher concentrations of P H I , that i s , when more than 40 fmol of PIII-DNA complex was retained by the f i l t e r s , the binding curves for UV-DNA and AAAF-DNA started to l e v e l o f f . We have estimated the u n i t s of DNA-binding a c t i v i t y of the various aliquots of P H I from the l i n e a r p ortion of the binding curves. The l e v e l i n g o f f of the binding curves might be due to (1) a l i m i t e d binding capacity of the f i l t e r s ; (2) a low retention e f f i c i e n c y 34 30 20 0 E L Q) •P C 0 TJ d) C (D •P Q) L < 2 • 10 / F 30 20 ± ± ± 1000 2000 U V d o s e , J / r r . 2 10 V 0.01 0.02 A A A F d o s e , m g / m l Fig. 7. DNA-binding of PHI as a function of UV-dose and AAAF-dose. The DNA-binding assays were performed under the standard conditions with an aliquot of PHI containing 23.5 units of UV-DNA-binding activity. 20 40 60 P l l l . p l Fig. 8. DNA-binding as a function of the amount of PHI. The DNA-binding assays were performed under the standard conditions with various amounts of PHI. PHI had a UV-DNA-binding activity of 2.5 units/yl. The assays were performed with UV-DNA ( • ) , AAAF-DNA (•) or u-DNA (O) as the binding substrates. Each point i s the average of duplicate assays. 36 of the PIII-DNA complex; or (3) a limited number of DNA-binding sites. In order to rule out possibility (1), two reaction mixtures containing PHI with 100 units of UV-DNA-binding activity were f i l t e r e d through the same glass fibre f i l t e r . The amount of DNA retained was about 140 fmol of DNA, which was twice the amount retained when a single reaction mixture was fi l t e r e d (Fig. 8). Thus the binding capacity of the f i l t e r was not limiting. The experiment also suggested that washing the f i l t e r with an additional 3.7 ml of f i l t r a t i o n buffer (2 ml from the second assay mixture and 1.7 ml from washing the second reaction tube) did not result in a significant elution of the PIII-DNA complex retained during the f i r s t f i l t r a t i o n . The retention efficiency of the glass fibre f i l t e r s was determined by f i l t e r i n g a reaction mixture through three f i l t e r s stacked on top of each other. PIII-DNA complex was only detected on the f i r s t f i l t e r , and was not present in the f i l t r a t e which passed through the f i r s t f i l t e r (Table III). Assuming that interaction with the f i l t e r does not cause the dissociation of the PIII-DNA complex, the retention efficiency of the f i l t e r i s close to 100%. The formation of complex between PHI and UV- or AAAF-DNA was largely due to the binding of PHI to UV- or AAAF-induced sites on the DNA (specific binding of PHI to UV- or AAAF-DNA) . However with a saturating amount of PHI, a significant amount of complex i s expected to be formed as a result of the binding of PHI to sites that are not induced by DNA damage. To estimate the amount of specific binding to UV- or AAAF-DNA for a saturating amount of PHI, the data points i n Fig. 8 were each corrected by using the equation: Table III. Efficiency of retention of PIII-DNA complexes by the GF/C f i l t e r s . DNA retained on f i l t e r s , fmol uv-: DNA u--DNA Experiment no. of f i l t e r +PIII -PHI +PIII -PHI I 1st 33.0 1.0 3.5 1.4 2nd 1.3 0.8 6.7 1.4 3rd 0.6 0.7 0.6 0.8 II 1st 30.8 1.3 4.4 0.7 In experiment I, each DNA-binding assay mixture was filtered through three f i l t e r s stacked on top of each other. In experiment II, each assay mixture was fi l t e r e d through one f i l t e r . The data are averages of duplicate assays. 38 y = A / 0 - 8 - B / 0 - 8 x 100% (1) 100% - B/0.8 where y i s the % specific retention of supercoiled FM2 DNA that i s either UV-irradiated or AAAF-treated; and A and B are the % retention of UV- or AAAF-DNA and u-DNA, respectively. The factor of 0.8 takes into account that only 80% of the PM2 DNA were supercoiled. As w i l l be discussed in a later section, PHI did not bind e f f i c i e n t l y to the nonsupercoiled form of UV- or AAAF-DNA. The corrected binding curve i s shown i n Fig. 9. A plateau level of DNA retention i s approached when the amount of PHI in the assay mixtures exceeds 100 units of UV-DNA-binding activity. The plateau corresponds to a retention of about 55% of the supercoiled UV- or AAAF-DNA. It should be noted that equation (1) i s only valid for a saturating amount of PHI. For a nonsaturating amount of PHI, i t leads to an overestimation of the amount of specific binding of PHI to UV- or AAAF-DNA since the binding a f f i n i t y of PHI to UV- or AAAF-DNA i s about 30-fold higher than u-DNA (see later section entitled "Substrate s p e c i f i c i t y " ) . Fig. 10 shows the binding curves for PHI determined at a DNA concentration 16-fold lower than that used in the standard assay conditions. The DNA was UV-irradiated with a dose of 1,200 J/m or 600 J/m . Again, the data points i n Fig. 10 are corrected by using equation (1) to plot the specific binding curves shown in Fig. 11. With saturating amounts of PHI, the specific binding curve for DNA UV-2 irradiated with a dose of 1,200 J/m approaches a plateau which corresponds to the retention of about 65% of the supercoiled PM2 39 F i g . 9. S p e c i f i c binding of UV-DNA and AAAF-DNA with various amounts of P H I . The data points f o r UV-DNA (•) or AAAF-DNA (•) were calculated from the r e s u l t s of F i g . 8 as described i n text. 40 P r o t e i n , J_I I Fig. 10. DNA-binding as a function of the amount of PHI at a low concentration of DNA substrates. The DNA-binding assays were performed under the standard conditions but with 8.7 fmol of PM2 DNA and with various amounts of PHI as indicated. The UV-DNA-binding activity of PHI used was 3.3 units/ul. DNA-binding activity was assayed with PM2 DNA UV-irradiated at 1200 J/m2 (#), PM2 DNA UV-irradiated at 600 J/m2 ( A ) and u-DNA (O ) . Each point is.the average of duplicate assays. 41 PI I I , Jjl Fig. 11. Specific binding of UV-DNA with various amounts of PHI at a low concentration of DNA substrates. The data points 2for DNA UV-irradiated with 1,200 J/m (• ) or 600 J/m ( A ) were calculated from the results shown in Fig. 10 as described i n text. 42 2 DNA. For DNA UV-irradiated with a lower dose of 600 J/m , a lower plateau value which corresponds to 45% of the supercoiled PM2 DNA i s obtained. These results can be interpreted as follows. After 2 2 irradiation of DNA at doses of 1,200 J/m and 600 J/m , about 65% and 45% of the supercoiled DNA contained at least one UV-induced binding sites for PHI, respectively. This interpretation is only valid i f every PIII-DNA complex formed in the assay mixture was retained by the f i l t e r and i f the binding of one PHI molecule was enough to cause the retention of a PM2 DNA molecule. The f i r s t assumption was already discussed in the previous paragraphs. The second assumption seems to be valid since DNA-binding i s linear at low concentration of PHI, where DNA i s in excess (Fig. 12). If the retention of DNA required more than one binding event, a sigmoidal binding curve would be expected (56). The linearity of the binding curve also indicated that the binding of PHI to DNA i s noncooperative. With higher UV-doses more PHI binding sites per DNA molecule can 2 be introduced. With a UV-dose of 3,600 J/m , about 70% of the total DNA (or 90% of the supercoiled DNA) was retained on the f i l t e r s (Fig. 13). Thus, the leveling off of the binding curves depicted in Fig. 8-11 was l i k e l y due to limited binding sites on the UV- or AAAF-DNA molecules. 4. Substrate specificity Fig. 14 illustrates a competition experiment where increasing amounts of unlabeled UV- or u-DNA were added to the reaction mixture to compete with a constant amount of labeled AAAF-DNA. With a 32-fold excess of UV-DNA i n the assay mixture, the binding of PHI to AAAF-DNA was nearly eliminated. This suggested that PHI i s a single protein 43 Fig. 12. DNA-binding curve at low concentration of PHI. The DNA-binding assays were performed under the standard conditions with various amounts of PHI. PHI used has a UV-DNA-binding activity of 2.5 units/ul. The binding substrates were UV-DNA (•) or u-DNA ( O ) . Each point i s the average of duplicate assays. 44 L (1) C 0 80 60 TJ QJ £ 40 CD Q) L < 20 z • / U V d o s e , J / m 2 x 10" 3 F i g . 13. Binding of P H I to DNA i r r a d i a t e d with high UV-doses . The DNA-binding assays were performed under the standard conditions with 8.7 fmol of UV-irradiated PM2 DNA. Each assay was c a r r i e d out with an al i q u o t of P H I containing 66 u n i t s of UV-DNA-binding a c t i v i t y . ) 45 I l | I I I I 1 L _ 4 8 12 16 20 24 28 32 R a t i o o f c o m p e t i t o r D N A t o A A A F D N A Fig. 14. AAAF-DNA-binding activity of PHI in the presence of competitor DNA. 3H-AAAF-DNA binding activity of PHI was assayed under the standard conditions in the presence of various amounts of unlabeled UV-DNA (•) or u-DNA (O) . which binds to both UV-DNA and AAAF-DNA. PHI binds less e f f i c i e n t l y to u-DNA since a 32-fold excess of u-DNA reduces the AAAF-DNA-binding activity by 50%. A reciprocal plot of the data of the competition experiment was made according to Spillman et a l . (58) to determine the relative binding a f f i n i t y of PHI to the different DNA substrates (Fig. 15). With UV-DNA as the competitor, the reciprocal plot yields a straight line with a slope close to 1, indicating that PHI binds to UV-DNA and AAAF-DNA with the same a f f i n i t y . With u-DNA as the competitor, the reciprocal plot yields a straight line with a slope of about 0.03. Thus, PHI has about 30-fold less binding a f f i n i t y to u-DNA than UV- or AAAF-DNA. Since the average number of binding sites per DNA molecule on the AAAF-DNA and UV-DNA are about equal (Fig. 9), we can conclude that PHI has the same af f i n i t y towards the AAAF-induced and UV-induced DNA-binding sites. To investigate the effect of DNA conformation on the binding activity of PHI, supercoiled AAAF-DNA, UV-DNA and u-DNA were nicked with bovine pancreatic DNase I or converted to a linear form by treatment with the restriction endonuclease Msp I. Msp I i s an isoschizomer of Hpa II. Both restriction enzymes recognize the sequence CCGG. However, in contrast to Hpa II, Msp I cleaves the DNA even i f the internal cytosine i s methylated (59, 60). Hpa II makes only one double-stranded cut per PM2 DNA molecule (61). Sucrose gradient analyses of PM2 DNA cleaved by Msp I indicated that most of the circular form of PM2 DNA was converted to the linear form (Fig. 16). The linear form of PM2 DNA sedimented slightly slower than the nicked circular form of PM2 DNA. This i s analogous to the observation that the linear HF III form of <j>X174 phage DNA has a smaller S value than the 47 / 1 1 I I I I I L_ 4 8 12 16 20 24 28 32 R a t i o o f c o m p e t i t o r D N A t o A A A F D N A Fig. 15. A reciprocal plot of the data of the competition experiment depicted in Fig. 14. x i s the ratio of the amount of AAAF-DNA retained on a f i l t e r in the presence of competitor DNA to the amount of AAAF-DNA retained in the absence of competitor DNA. Unlabeled UV-DNA (•) or u-DNA (O) was used as competitors. 48 Fig. 16. Sucrose gradient sedimentation of Msp I-treated DNA. FM2 DNA was treated with Msp I (open symbols) or without Msp I ( f i l l e d symbols). The DNA then was subjected to sucrose gradient sedimentation as described in Materials and Methods. (A) DNA which was UV-irradiated with 1200 J/m2 ( • , O). (B) DNA which was treated with 0.02 mg/ml of AAAF ( • , • ) . (C) untreated DNA ( A , A). The sedimentation was from right to l e f t . B Fig. 16 (B). c Fig. 16 (C). nicked circular RF II form (62). It i s clear from Table IV that PHI prefers supercoiled DNA as the binding substrate. PHI binds to the linear or relaxed circular form of UV- or AAAF-DNA 5- to 10-fold less e f f i c i e n t l y than to the supercoiled form of DNA. We have also tested the binding activity of PHI towards DNA treated with two alkylating agents, MNNG and MMS. Using different types of filter-binding assays for DNA lesions, the extent of DNA alkylation can be estimated (49). The levels of alkylation in the various MMS-DNA preparations were similar to or greater than the two MNNG-DNA preparations (Table V) . The binding activity of PHI assayed with DNA treated with 1 mM MNNG was about 40% of that assayed with UV-or AAAF-DNA. DNA treated with 5 mM MNNG was at least as efficient a substrate for PHI as UV- or AAAF-DNA. On the other hand, DNA treated with 10-100 mM MMS had l i t t l e i f any binding sites for PHI (Table VI) . PHI did not bind effic i e n t l y to depurinated DNA with about 1.5 apurinic sites. The binding activity with single-stranded DNA as a substrate was the same as with u-DNA. The latter result suggests that the single-strandness of the DNA alone does not account for the DNA-binding activity of PHI. 5. Other properties of PHI DNA-binding activity of PHI was eliminated to a large extent after a treatment with 20 yg/ml of proteinase K at 37°C for 30 min. When incubation at 37°C was omitted, the DNA-binding reaction of PHI was not inhibited, indicating that proteinase K did not interfere with the formation of PIII-DNA complex (Experiment I and II, Table VII). We therefore concluded that the DNA-binding activity of PHI was due Table IV. Effect of DNA conformation on the DNA-binding activity of F i l l . DNA retained on f i l t e r , fmol Treatment AAAF-DNA UV-DNA u-DNA -Msp I 15.4 (100%) 15.4 (100%) 1.4 (9%) +Msp I 3.2 (21%) 1.7 (11%) 0.0 (0%) -DNase I — 14.9 (100%) 1.5 (10%) +DNase I — 3.5 (23%) 0.6 (4%) The DNA was treated with UV or AAAF prior to cleavage by Msp I or DNase I as described in Materials and Methods. The values in parentheses were % DNA-binding activity relative to that assayed with supercoiled UV-DNA. 53 Table V. Estimation of DNA damage on various DNA substrates. Average number of nicks (or filter-binding sites) per DNA molecule DNA Assay I Assay II Assay III Assay IV MNNG-DNA (5 mM) 0.74 1.58 0.88 3.10 (1 mM) 0.40 0.67 0.43 • 1.12 MMS-DNA (100 mM) 1.23 >3.50 1.67 >3.50 (40 mM) 0.43 2.40 0.85 >3.50 (20 mM) 0.35 1.10 0.62 >3.50 (10 mM) 0.25 0.57 0.36 1.90 u-DNA 0.22 0.20 0.17 0.24 AAAF-DNA 0.37 0.36 0.25 0.48 UV-DNA 0.27 0.34 0.29 0.66 Assay I is the standard nicking assay as described in Materials and Methods. It measures mainly single- and double-stranded breaks in DNA and to a certain extent local distortions of DNA. Assay II i s the standard nicking assay except that the DNA is incubated for 45 min at 37 C after addition of the a l k a l i denaturation buffer. Assays III and IV were the same as assays I and II, respectively, except that the DNA was incubated at 70 C for 7.5 min before the nicking assays were performed. The amount of alk a l i - l a b i l e sites in the DNA can be calculated by subtracting the results of assay I from those of assay II. The amount of heat-induced a l k a l i - l a b i l e sites can be estimated by subtracting the results of assay III and the amount of al k a l i - l a b i l e sites from those of assay IV. The amount of DNA breakage detected by the various assays generally increases with increasing level of DNA alkylation. For a discussion of the scope of the various types of DNA damage that can be measured with these assays and the limitations of the assays, see Kuhnlein et a l . (49). Table VI. Substrate s p e c i f i c i t y of P H I . Substrate % a c t i v i t y UV-DNA 100 AAAF-DNA 100 u-DNA 12 MNNG—DNA (5 mM) 97 (1 mM) 39 MMS-DNA (100 mM) 10 (40.mM) 15 (20 mM) 12 (10 mM) 10 depurinated DNA 18 single-stranded DNA 10 DNA-binding a c t i v i t i e s were assayed under the standard conditions with the various DNA substrates. 15-20 units (UV-DNA-binding a c t i v i t y ) of P H I were used i n these experiments. The data for UV-DNA, depurinated DNA and single-stranded DNA are the averages of duplicate assays. The various DNA substrates were prepared as described i n Materials and Methods. The binding a c t i v i t y towards single-stranded DNA has been m u l t i p l i e d by two since binding to one double-stranded DNA molecule gives twice the amount of r a d i o a c t i v i t y as the binding of one single-stranded DNA molecule. 55 Table VII. Sensitivity of PHI to proteinase K and RNase A treatments Experiment Treatment Incubation at 37°C, min DNA retained UV-DNA on f i l t e r , fmol u-DNA I -Proteinase K 30 14.2 2.8 +Proteinase K 30 0.3 1.0 II -Proteinase K 0 20.8 4.0 +Proteinase K 0 20.1 3.5 III -RNase A 30 7.6 1.1 +RNase A 30 7.3 0.8 Proteinase K solutions (1 mg/ml) were prepared in a buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and 1 mM CaCl This solution was further diluted to 0.2 mg/ml with 10 mM Tris-HCl, pH 7.5, and 0.1 M NaCl. Aliquots of PHI were incubated with 20 ug/ml of proteinase K or 100 ug/ml of RNase A at 37 C for various periods of time as indicated. The treated aliquots of PHI were then assayed for DNA binding ac t i v i t i e s under the standard conditions. to protein molecules. In addition, RNA appears not to be involved in the DNA-binding by PHI, since PHI i s not sensitive to RNase A (Experiment III, Table VII). The presence of 1-7 mM MgC^ or MnC^ i n the assay mixtures did not markedly affect the binding of PHI to UV-DNA and u-DNA. (Fig. 17). The binding activity of PHI was also measured with the assay mixture buffered with either Tris-HCl or potassium phosphate at different pH values. PHI bound to UV- or u-DNA to a similar extent at pH values between 6 and 9 (Fig. 18). Variation of the incubation temperature of the reaction mixture between 0°C and 37°C did not affect the DNA-binding activity of PHI significantly (Table VIII). The DNA-binding activity of PHI Is quite resistant to freeze-thaw treatment. In two separate experiments, less than 25% of the UV-DNA-binding activity of PHI was lost by freezing and thawing several times (Table IX). PHI i s also quite heat stable. When incubated for 50 min at 60°C in a buffer containing 5 mM Tris-HCl, pH 7.5^ 0.5 M NaCl, 0.5 mM EDTA, 0.5 mM DTT, 55% glycerol and 100 pg/ml of 3-lactoglobulin, PHI lost only 30-40% of i t s binding activity to UV-DNA (Fig. 19). The inactivation of the binding activity of PHI to u-DNA was faster, a loss of 70-80% of the binding activity was observed in 50 min at 6Q°C. This result suggests that the UV- or AAAF-DNA-binding activity of PHI may be contaminated with a protein (or proteins) which binds only to u-DNA. The latter may be purified from the former by glycerol gradient sedimentation (see later section and Fig. 23). Three chemicals were found to inhibit the DNA-binding activity 57 Fig. 17. Effect of MgCl ? and MnCl- on the binding activity of PHI. The DNA-binding assays were performed under the standard conditions i n the presence of various concentrations of MgCl 2 (#,0) or MnCl2 ( A , A ) in the assay mixtures. DNA-binding activity was assayed with UV-DNA ( • , A ) or u-DNA (O , A ) . 58 30h 0 E ii= 20 -C 0 TJ 03 C "to 6 7 8 9 p H Fig. 18. DNA-binding activity of PHI: pH dependence. DNA binding reactions were carried out under the standard conditions except that the reaction mixtures were buffered with 10 mM Tris-HCl (• ,0) or potassium phosphate ( A ,A) at the indicated pH values. Each reaction was performed with an aliquot of PHI containing about 25 units of UV-DNA-binding activity. After incubation for 10 min on ice, each assay mixture was diluted with 1.7 ml of 100 mM NaCl and fi l t e r e d under the standard conditions. The binding substrates were UV-DNA ( • , A ) or (O.A). The pH values of the buffers were measured at 50 mM salt concentration at room temperature (22°C). The pH values of the Tris-HCl buffers at 0 C w i l l be about 0.7 higher (Trizma, Sigma Technical Bulletin No. 106B). Table VIII. Effect of temperature on the DNA-binding activity of PHI. DNA retained on f i l t e r , fmol Temperature (°C) UV-DNA u-DNA 0 23.3 3.5 22 20.4 2.8 37 23.2 3.9 The DNA-binding assays were carried out under the standard conditions except that the assay mixtures were incubated at various temperatures as indicated. The data are averages of duplicate assays. Table IX. Freeze-thaw s t a b i l i t y of P H I . DNA retained on f i l t e r , fmol Freeze-thaw Experiment treatment UV-DNA u-DNA I - 25.A 2.8 + 19.6 2.5 II - 7.1 0.8 + 5.4 0.8 Ali q u o t s of P H I were frozen i n l i q u i d nitrogen and thawed with cold running water. The freeze-thaw procedure was repeated f i v e times i n experiment I and three times i n experiment I I . The freeze-thaw treated a l i q u o t s and untreated a l i q u o t s of P H I were then assayed for DNA-binding a c t i v i t y . The data are the averages of duplicate assays. 61 20 m i n a t B O C 40 F i g . 19. Heat s e n s i t i v i t y of P H I . Aliq u o t s of P H I were heated at 60 C for various periods of time and then assayed for DNA-binding a c t i v i t y with UV-DNA (•) or u-DNA ( O ) as the binding substrates. I n i t i a l l y , each aliq u o t of P H I contained about 21 uni t s of UV-DNA-binding a c t i v i t y per r e a c t i o n mixture. 62 of PHI to UV-DNA and u-DNA. They were ATP, caffeine and sucrose. In the presence of 10 mM ATP, the binding of PHI to UV-DNA was reduced by 40% and the binding of PHI to u-DNA was reduced to nearly zero CFig. 20). In thisjegard, PHI is different from the uvrA protein of Escherichia coli whose binding activity to UV-DNA i s stimulated by the presence of ATP and GTP. Caffeine, which i s known to inhibit DNA repair and bind to single-stranded regions of DNA (63, 64), is an inhibitor of PHI binding (Fig. 21). The inhibitory effect of caffeine was greater with AAAF-DNA and u-DNA than with UV-DNA. 3 mM caffeine inhibited the UV-DNA-binding activity of PHI by about 35%, the AAAF-DNA-binding activity by nearly 70% and the u-DNA-binding activity by about 60%. In the experiment shown in Fig. 21, the DNA was preincubated with caffeine for 30 min before the addition of PHI. Similar results were obtained in experiments where neither PHI nor DNA were preincubated with caffeine or where PHI rather than the DNA was preincubated with caffeine for 30 min. It i s l i k e l y that caffeine binds to DNA and thereby alters or masks the DNA-binding sites for PHI. This i s suggested by the observation of a differential effect of caffeine on the binding of PHI to AAAF-DNA and UV-DNA. The binding activity of PHI was also inhibited by sucrose. The presence of 5% sucrose in the assay mixture reduced the UV-DNA-binding activity by half and the u-DNA-binding activity to nearly zero. On the other hand, glycerol had l i t t l e effect on the DNA-binding activity of PHI (Table X). Based on these observations, sedimentation experiments were performed with glycerol gradients rather than sucrose gradients. F i g . 20. E f f e c t of ATP on the DNA-binding a c t i v i t y of P H I . DNA-binding assays were c a r r i e d out under the standard conditions i n the presence of various concentrations of ATP. The binding substrates were UV-DNA (•) or u-DNA ( O ) . Each point i s the the average of duplicate assays. 25f C a f f e i n e , m M Fig. 21. Effect of caffeine on the binding activity of PHI. Caffeine at the indicated concentrations was added to the standard assay mixture and incubated with UV-DNA (• ) , AAAF-DNA (•) or u-DNA (O) for 30 min on ice. Afterwards, PHI was added and the DNA-binding assays were carried out under the standard conditions. Table X. Effects of sucrose and glycerol on the DNA-binding activity of PHI. DNA retained on UV-DNA f i l t e r , fmol u-DNA % sucrose 0 12.2 2.4 5 6.9 0.3 10 2.6 0.6 20 0.7 0.8 % glycerol 10 13.6 1.4 30 11.5 1.7 The binding assays conditions except various amounts of were carried out under that the assay mixtures sucrose or glycerol as the standard also contained indicated. The experiments described above were carried out at a saturating level of DNA damage. At nonsaturating level of DNA damage, the dependence of the DNA-binding activity of PHI on NaCl, temperature, MgCl 2, MnCl 2 or pH was similar. However, the inhibitory effect of ATP on the binding activity of PHI to UV-DNA or u-DNA was more pronounced and am ounted to a 60% inhibition at 3 mM ATP when the DNA-binding assays were carried out with DNA UV-irradiated with 580 J/m2 and aliquots of PHI each containing 34 units of UV-DNA-binding activity. 6. Glycerol gradient sedimentation analysis of PHI In order to get an estimate of the molecular weight of the DNA-binding protein, aliquots of PHI were analysed by sedimentation in 10-30% glycerol gradients. 100 yg/ml of bacitracin was included in the glycerol gradient solution to stabilize the DNA-binding activity of PHI during the sedimentation. Bacitracin i s an antibiotic produced by Bacillus lichenifovmis. It consists of a mixture of closely related polypeptides. The major component i s a cyclic dodecapeptide called bacitracin A with a molecular weight of about 1,500 (65). Bacitracin was chosen because of i t s low molecular weight. A preliminary experiment Indicated that the presence of 100 yg/ml of bacitracin in PHI did not affect the DNA-binding activity of PHI. Fig. 22 shows that PHI sedimented through a 10-30% glycerol gradient containing 0.15 M NaCl with a sedimentation coefficient of 2.0-2.5 S relative to the marker proteins. A similar sedimentation coefficient was obtained when the centrifugation was carried out in 0.5 M NaCl in the gradient (Fig. 23). The ratio of the AAAF- or UV-DNA-67 Fig. 22. Sedimentation velocity analysis of PHI in the presence of 0.15 M NaCl. A 200-yl aliquot of PHI in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 3.3% glycerol, 0.3 M NaCl and 100 yg/ml of 8-lactoglobulin was sedimented for 17 h through a 10-30% glycerol gradient containing 0.15 M NaCl as described in Materials and Methods. 37 fractions were collected. A 50-ul aliquot from each fraction was assayed for binding activity towards UV-DNA ( • ) or u-DNA ( O ) . Marker proteins were: A, ovalbumin; B, cytochrome C. The sedimentation was from right to l e f t . 68 D E L P c 0 TJ Q) C CD P CD c_ < Z • 20 40 F r a c t i o n Fig. 23. Sedimentation velocity analysis of PHI in the presence of 0.5 M NaCl. A 200-ul aliquot of PHI in 10 mil Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 5% glycerol, 0.5 M NaCl and 100 ug/ml of B-lactoglobulin was sedimented for 27 h through a 10-30% linear glycerol gradient containing 0.5 M NaCl as described i n Materials and Methods. 35 fractions were collected. A 30-yl aliquot from each fraction was assayed for binding activity towards UV-DNA (• ) , AAAF-DNA (•') or u-DNA ( o ) . Marker proteins were: C, bovine serum albumin; D, a-chymotrypsin; and E, myoglobin. The sedimentation was from right to l e f t . binding activity to the u-DNA binding activity of PHI increased from 10:1 in the former experiment to 20:1 i n the latter experiment, indicating a further purification of the specific binding activity for AAAF-DNA and UV-DNA. Assuming the protein i s spherical, the molecular weight of PHI was estimated to be 20-25,000. The recovery of the DNA-binding activity from these glycerol gradients was greater than 90%. 7. Characterisation of the PIII-DNA complex Glass fibre f i l t e r s are commonly used to retain protein or DNA precipitates. We therefore investigated the possibility of DNA precipitation in our assays. An aliquot of PHI containing 60 units of UV-DNA-binding activity was incubated with UV-DNA under the standard conditions. The assay mixture was then centrifuged for 10 min at- 10,000 g. It was found that a l l the DNA remained in solution. PIII-DNA complex was also analysed by sedimentation through 10-30% glycerol gradients in the presence of 0 mM NaCl, 50 mM NaCl and 150 mM NaCl. Two major forms of PM2 DNA were resolved by velocity gradient sedimentation: the faster sedimenting covalently-closed circular supercoiled form and the nicked, relaxed circular form. Incubation with PHI did not induce any detectable changes in the sedimentation profiles of UV-DNA (Fig. 24-26). PHI bound primarily to the supercoiled form of UV-DNA but had much less binding activity towards the nicked form of PM2 DNA (Inserts, Fig. 24-26). In the absence of NaCl, there was very l i t t l e i f any dissociation of the bound PHI from the supercoiled DNA (Insert, Fig. 24). In the presence of 50 mM NaCl, half of the bound PHI molecules dissociated from the UV-DNA during the 2 h of sedimentation (Insert, Fig. 25); and i n the presence 70 el CN I o E a o % uv--DNA retained on f i l t e r + PHI • PHI 56.6 (14-17) 0.4 (14-17) 5.4 (20-22) 1.9 (20-22) J v" \ fr 10 20 30 F r a c t i o n Fig. 24. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol and 0 mM NaCl. UV-DNA was incubated with an aliquot of PHI containing 80 units of UV-DNA-binding activity (A ) or without PHI (A) under the standard conditions. 200 ul of the assay mixture was sedimented through a 10-30% glycerol gradient in the absence of NaCl for 3 h as described in Materials and Methods. The sedimentation was from right to l e f t . In the experiment where the UV-DNA was incubated with PHI, 35 fractions were collected. In the experiment where the UV-DNA was incubated without PHI, 34 fractions were collected. A 50-ul aliquot from each fraction was was assayed for radioactivity. The background (25 cpm) was not subtracted. The remaining portions of the peak fractions were f i l t e r e d over GF/C f i l t e r s to measure the amount of PIII-DNA complexes and the results were shown in the insert. Numbers in parentheses in the insert indicated the fraction numbers. 71 % uv--DNA retained on f i l t e r + PHI PHI 19.6 (20-23) -0.2 (20-23) 3.8 (24-26) 0.5 (24-26) Fig. 25. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol and 50 mM NaCl. The experiment was performed as described in the legend to Fig. 24 except that the glycerol gradient solution contained 50 mM NaCl i n addition to the other ingredients and the sedimentation was for 2 h. 34 fractions were collected for each sedimentation analysis. 72 8 CN I o X E a u A J l % uv--DNA retained on f i l t e r + PHI PHI 2.5 (18-21) -0.2 (17-20) 1.6 (23-25) 2.2 (21-23) \A A 10 20 F r a c t i o n , + P I 30 10 20 F r a c t i o n . — P I 30 Fig. 26. Sedimentation of PIII-UV-DNA complex in 10-30% glycerol and 150 mM NaCl. The experiment was performed as described in the legend to Fig. 24 except that the glycerol gradient solution contained 150 mM NaCl in addition to the other ingradients and the centrifugation was for 2 h. In the experiment where UV-DNA was incubated with PHI ( A ) , 36 fractions were collected. In the experiment where the UV-DNA was incubated without PHI ( A ) , 34 fractions were collected. To f a c i l i a t e comparison of the two sedimentation profiles, the fraction numbers of the latter experiment were plotted on a different scale. of 150 mM NaCl, the d i s s o c i a t i o n was nearly complete i n 2 h (Insert, F i g . 26). The e f f e c t of s a l t on the d i s s o c i a t i o n of P H I from nicked DNA was q u a l i t a t i v e l y s i m i l a r . These r e s u l t s do not contradict the f i n d i n g that the s p e c i f i c binding of P H I to UV-DNA was optimal between 100-200 mM NaCl as i l l u s t r a t e d i n F i g . 3. Sedimentation separated the free molecules of P H I from the DNA and thereby favored the d i s s o c i a t i o n of PIII-DNA complex. Thus the binding of P H I to UV-DNA was r e v e r s i b l e and did not involve the formation of covalent bonds between P H I and DNA. Covalent bonds between proteins and DNA are formed by adenovirus terminal-protein (43, 44), Escherichia coli topoisomerase I (66) and the DNA untwisting enzyme of rat l i v e r (67). PIII-DNA complex was also analysed for undamaged DNA i n the absence of NaCl. Again, the binding of P H I did not a l t e r the sedimentation p r o f i l e of the DNA (F i g . 27) and there was a strong binding preference for supercoiled DNA (Insert, F i g . 27). Sedimentation analyses for PIII-u-DNA complex i n the presence of NaCl was not performed. Incubation of UV-DNA with P H I for 45 min at 37°C i n the presence of 3 mM ATP, 7.5 mM MgCl 2 and 1 mM DTT also did not induce any change i n the sedimentation p r o f i l e of the DNA (Fig. 28), nor did i t render the complex more stable (Insert, F i g . 28). The r e v e r s i b i l i t y of the binding of P H I to UV-DNA was also evident from the competition experiment shown i n F i g . 29. The r e s u l t s i n d i c a t e that P H I dis s o c i a t e s from the UV-DNA very slowly a f t e r i t i s bound. The amount of the ra d i o a c t i v e PIII-UV-DNA complex was only reduced by % u-DNA retained on f i l t e r + PHI -PHI 24.2 (13-17) 1.0 (13-17) 4.4 (19-21) 2.9 (19-21) 10 20 F r a c t i o n 30 Fig. 27. Sedimentation of PIII-u-DNA complex in 10-30% glycerol and 0 mM NaCl. The experiment was performed as described In the legend to Fig. 24 with u-DNA incubated with (• ) or without PHI ( • ) . 34 fractions were collected for each sedimentation analysis. 75 A » % uv--DNA retained on f i l t e r + P H I - P H I 18.3 (19-22) 0.1 (19-22) 4.2 (23-25) 2.5 (23-25) A 1/ % #X 2 30 F r a c t i o n F i g . 28. Sedimentation of PIII-UV-DNA complex formed i n the presence of ATP and MgCl2« 139 fmol fo UV-DNA was incubated with 80 un i t s (UV-DNA binding a c t i v i t y ) of P H I ( A ) or without P H I ( A ) i n 300-ul of buffer containing 10 mM Tris - H C l (pH 7.5), 3 mM ATP, 7.5 mM MgCl 2 and 1 mM DTT. Aft e r incubation f or 45 min at 37°C, a 200-yl a l i q o u t was subjected to sedimentation analysis as described i n the legend to F i g . 25. 33 f r a c t i o n s were c o l l e c t e d f o r each sedimentation analyses. 76 Fig. 29. Reversibility of the binding of PHI to UV-DNA. Aliquots of PHI were incubated with labeled UV-DNA under the standard conditions. Prior to f i l t r a t i o n , the assay mixtures were further incubated for various periods of time with 5 y l of 10 mM Tris-HCl, pH 7.5, (•) or 5 y l of 10 mM Tris-HCl, pH 7.5, containing 1.39 pmol of unlabeled PM2 DNA. The unlabeled DNA was either unirradiated (O) or UV-irradiated with 1200 J/m2 (•). Each point i s the average of duplicate assays. 40% when incubated with a 10-fold excess of unlabeled UV-DNA at 0UC for 30 min. Assuming f i r s t order kinetics, the dissociation can be described by the equation: - In | = k 2 t (2) o where ^ i s the rate constant for the dissociation of the PIII-UV-DNA complex; t i s the time of incubation; B q is the amount of PIII-DNA complex at time 0; and B i s the amount of complex at time t. The -4 -1 estimate for k^ i s about 3 x 10 sec , which corresponds to a L h a l f - l i f e of the complex of about 40 min. 8. Catalytic activity No significant amount of endonuclease or glycosylase activity was detected in aliquots of PHI under the various conditions described in Tables XI and XII. Incubation of UV- or u-DNA under the standard conditions with aliquots of PHI containing 80 units of UV-DNA-binding activity did not produce any material soluble i n 6% trichloroacetic acid. UV-DNA, u-DNA or linear PM2 DNA which had been incubated with PHI for 45 min at 37°C i n the presence of 10 mM Tris-HCl, pH 7.5, 33 mM NaCl and 5 mM MgC^ also was not soluble i n 6% trichloroacetic acid. Thus, under these two conditions, PHI does not exhibit any exonuclease activity or behave like certain glycoproteins which solubilize DNA in dilute trichloroacetic acid without degrading the DNA (68, 69). No ATPase activity was detected with aliquots of PHI containing 50 units of UV-DNA-binding activity in the presence of UV-DNA, u-DNA or single-stranded PM2 DNA. Table XI. Assay for DNA endonuclease activity. % DNA nicked UV-DNA u-DNA +PIII 21.7 19.3 -PHI 19.7 18.8 Aliquots of 40 units (UV-DNA-binding activity) of PHI were incubated for 30 min at 37 C with 139 fmol of UV-DNA or u-DNA in a 300 y l of reaction mixture containing 10 mM Tris-HCl, pH 7.5, 33 mM NaCl and 5 mM MgCl 2. Afterwards, assays for DNA endonuclease activity were performed as described in Materials and Methods. The data are the averages of duplicate assays. Table XII. Assays for UV-DNA endonuclease and glycosylase act i v i t i e s under various conditions. % UV-DNA nicked MgCl 2 DTT ATP endonuclease assay glycosylase assay mM + PHI PHI + PHI PHI 0 0 0 28.2 28.9 47.9 48.3 0 0.1 0 30.5 29.9 43.1 44.0 2 0 0 30.7 30.1 42.1 42.3 2 0.1 0 30.6 30.0 41.4 40.3 5 0.1 0 30.7 29.5 5 0.1 3 29.8 30.0 Aliquots of PHI each containing 40 units of UV-DNA-binding activity were incubated with 139 fmol of UV-DNA or u-DNA in 300-ul reaction mixtures containing 10 mM Tris-HCl, pH 7.5, 33 mM NaCl and various concentrations of MgCl 2, DTT and ATP as indicated. Incubations were at 37 C for 30 min. Afterwards, 50-yl aliquots were assayed for DNA nicking or a l k a l i - l a b i l e sites as described in Materials and Methods. The amounts of DNA nicking and al k a l i - l a b i l e sites introduced by PHI were taken as a measure of the endonuclease and glycosylase a c t i v i t i e s , respectively. The data are the averages of duplicate assays. It i s possible that PHI may unwind the DNA double helix adjacent to the DNA binding site. We have therefore studied the effect of PHI on the susceptibility of UV-DNA and u-DNA to cleavage by the single-stranded specific endonuclease from Neurospora crassa. This endonuclease attacks supercoiled circular DNA, probably by recognizing some unpaired regions in the DNA (62, 70, 71). It preferentially nicked UV-irradiated DNA rather than unirradiated DNA (72). It has been shown that the destabilization of the DNA double helix by the rep protein of Escherichia coli f a c i l i a t e s cleavage of the DNA by the Neurospora crassa endonuclease (73). Fig. 30 shows that the binding of PHI did not alter the susceptibility of UV- or u-DNA to cleavage by the Neurospora crassa endonuclease. It also appears that the cleavage sites for the endonuclease are not masked by PHI. In our experiment,, the rate of nicking of the UV-DNA by the endonuclease i s 3-4 fold higher than that of u-DNA. 9. DNA-binding proteins in normal human and XP-fibroblasts DNA-binding proteins were also analysed in extracts of a normal human fibroblast and two XP-fibroblast c e l l lines. DEAE-fractions were prepared from about 5-6 x 10^ ce l l s and contained 9-13 mg of protein (Table XIII). When the DEAE-fraction of normal human fibroblasts was subjected to phosphocellulose chromatography, three major peaks of DNA-binding activity were eluted from the column as with Hela cells (Fig. 31). The peak which showed a binding preference for UV-DNA and AAAF-DNA rather than u-DNA was eluted at about 375 mM potassium phosphate. A similar elution profile was obtained with fibroblast extract of the XP-group D c e l l line, XP2NE. Fibroblasts of the XP-group A c e l l line, 81 0.5 1 u n i t s / m l Fig. 30. Effect of PHI on the susceptibility of DNA to the single-stranded specific endonuclease from Neurospora orassa. UV-DNA ( f i l l e d symbols) or u-DNA (open symbols) were incubated with 40 units (UV-DNA-binding activity) of PHI ( A , A ) o r without PHI ( • , O ) under the standard conditions except that the assay mixtures also contained 100 ug/ml of acetylated BSA. The assay mixtures were then incubated for 30 min at 37 C with 5-yl aliquots of various concentrations of the single-stranded specific endonuclease from Neurospora orassa. The f i n a l concentrations of the endonuclease were as indicated. 50-yl aliquots of the reaction mixtures were assayed for DNA nicking. Each point i s the average of duplicate assays. The Neurospora orassa endonuclease had an activity of 535 units/mg and was in 3.2 M (NHi^SOit, pH 6.0. It was diluted i n 10 mM Tris-HCl (pH 7.5) and 100 pg/ml of acetylated BSA prior to experiment. 82 Table XIII. Preparation of extracts used for the analyses of the DNA-binding proteins from human fibroblasts. Volume of Amount of protein in DEAE-fraction DEAE-fraction Cell line Cell number ml mg 2071 4.8 x 10 7 7.1 8.5 XP5EG2 5.1 x 10 7 7.1 9.2 XP2NE3 6.0 x 10 8.5 12.9 XP5EG(2)'t 5.0 x 10 7 8.0 9.4 A normal human c e l l line. A XP-group A c e l l line. A XP-group D c e l l line. A repeated analysis with the c e l l line XP5EG. Read legend of Fig. 31 for details. 60 E C 3 5 A < Poo, / 1 c o 0.A XI0.2 ca 4-1 c 01 u c o u 4J iH CO m 20 AO Fraction 60 Fig. 31. Phosphocellulose chromatography of DNA-binding proteins from human fibroblast extracts. Fibroblast extracts of the c e l l lines (A) 207, (B) XP5EG and (C) XP2NE were subjected to analyses. The extracts were from cells harvested at the 10th c e l l passage. (D) A repeated analysis for the c e l l line XP5EG was performed with cells harvested at the 13th passage. Each column fraction was assayed with either UV-DNA (• ) or u-DNA ( o ) under the standard conditions. Phosphate concentration ( ••••). o o Salt concentration, M XP5EG, seemed to be deficient i n a DNA-binding protein which eluted with 180-250 mM potassium phosphate from the column. A repeated analysis with an independent extract of XP5EG indicates that this deficiency was reproducible. The amounts of DNA-binding protein (expressed as units of DNA-binding activity per mg of protein i n the DEAE-fraction) which eluted at 375 mM potassium phosphate in the normal human fibroblasts and XP fibroblasts were similar to that of the phosphocellulose fraction of PHI from Hela c e l l s (Table XTV). 10. Estimation of the equilibrium constant of the binding reaction and the concentration of PHI For the estimation of the equilibrium constant of the binding reaction and the concentration of PHI, we w i l l assume that one PHI molecule i s enough to cause the retention of a DNA molecule and that every PIII-DNA complex i s retained by the f i l t e r . The valid i t y of these two assumptions was already discussed in an earlier section. We w i l l further assume that the binding reaction between PHI and the binding sites on the UV-DNA are as follows: The d i s s o c i a t i o n equilibrium constant of the re a c t i o n can be calculated according to the equation: P + ± PS [P f] [ s f ] (3) [PS] where P^ i s the concentration of the free PHI molecules, S,. i s the concentration of free binding sites on UV-DNA, and PS is the concentration of PIII-DNA complex. From equation (3), i t can be shown that [ S ] 1 / 2 = K d + 1/2 [P] (4) Table XIV. Summary of the analyses of a UV-DNA-binding pr o t e i n i n human f i b r o b l a s t e x t r acts. Units of a c t i v i t y x 10 per mg of protein i n the DEAE-fraction F r a c t i o n C e l l l i n e UV-DNA u-DNA DEAE 207 2.06 1.29 XP2NE 2.12 1.38 XP5EG 1.74 0.74 XP5EG(2) 1.90 0.70 Phospho- 207 0.96 0.21 c e l l u l o s e ^ XP2NE 0.82 0.10 XP5EG 0.88 0.10 XP5EG(2) 1.02 0.14 See text and legend of Table XIII f o r d e t a i l s of the various c e l l l i n e s . Peak f r a c t i o n s of the DNA-binding a c t i v i t y which eluted between 350-400 mM phosphate were pooled"and assayed i n duplicates f o r DNA-binding a c t i v i t y . where [ S ] - ^ i s t h e concentration of binding sites on UV-DNA that w i l l saturate one-half of the PHI molecules, and [P] i s the concentration of of PHI i n the assay mixture (56). As shown in Fig. 7, [S]^^2 2 occurs at an UV-dose of about 250 J/m . From Fig. 11, i t can be inferred that 50% of the supercoiled PM2 DNA UV-irradiated with a dose 2 of 600 J/m has at least one binding site for PHI. Assuming a Poisson distribution of the binding sites on the supercoiled DNA and that the average number of binding sites per supercoiled DNA molecule increases 2 linearly with an UV-dose up to 600 J/m , we calculate that the average number of binding sites per molecule of supercoiled DNA irradiated with an UV-dose of 250 J/m2 i s 0.29. With 3.7 x 10~ 1 0 M of supercoiled DNA molecule in the assay mixture, [ S ] ^ ^ i s therefore about 1.1 x 10~ 1 0 M. From the plateau of the dose response curve shown in Fig. 7, one can estimate that the assay mixture contained about 23.5 fmol of PHI molecules. [P] i s therefore 7.8 x 1 0 - 1 1 M. Using the values of t S ] ^ ^ [F]» i s calculated from equation (A) to be about 7 x 10 ^ M, which corresponds to a binding energy of 13 kcal/mol. Since the dissociation rate constant of the -A -1 binding reaction i s 2.9 x 10 sec as has been estimated from Fig. 30, the association rate constant of the binding reaction can be calculated to be A.l x 10^ M ^ sec \ The of the binding reaction of PHI i s about 2-3 orders of magnitude higher than the Kj for the binding of lac repressor to -13 operator which i s i n the order of 10 M (56). It i s much lower than the Kj values for the binding of lac repressor to nonoperator DNA which i s i n the order of 10 ^ M (7A), the noncooperative binding of gene 32 protein of phage T4 to duplex DNA which i s in the order of -4 10 M (75) and the noncooperative binding of the gene D5 protein of phage T5 to single-stranded DNA which i s 1.85 x 10~ 8 M (76). It i s , however, comparable to the Kj values of the cooperative binding of gene 32 protein to single-stranded DNA which i s in the order of 10~ 1 0 M (75) and the cooperative binding of the gene D5 protein to duplex DNA which i s '6.27 x 10 _ 1° M (76) We had used 10 y l of PHI in the experiment depicted i n Fig. 7. The results of the experiment suggests that there were 23.5 fmol of PHI molecules per 10 y l of PHI. The concentration of PHI was _9 therefore 2.35 x 10 M. If our purification scheme had a 30% recovery for PHI, and i f a l l PHI molecules were extracted from the Hela c e l l s , the amount of PHI per Hela c e l l was in the order of 10^ molecules. Discussion i . Advantages of using glass fibre f i l t e r s in the filter-binding assay We have used GF/C f i l t e r s in the filter-binding assay of PHI. Conventionally, nitrocellulose membrane f i l t e r s are used for the f i l t e r -binding assay of nucleic acid-binding protein (35-42, 56, 57, 77-81). For our present study, the nitrocellulose f i l t e r s have the disadvantage that they also bind single-stranded DNA and to a certain extent DNA with helical distortions (49, 82). To reduce a high background due to the retention of these forms of DNA in the filter-binding assays, i t i s necessary to treat the nitrocellulose f i l t e r s in an a l k a l i solution followed by extensive washing and neutralization of the f i l t e r s (42, 77, 80, 81). Glass fibre f i l t e r , however, retained less than 2% of the various kinds of DNA we have used i n our present study, including the single-stranded DNA. In comparison to the nitrocellulose f i l t e r s , glass fibre f i l t e r s are more convenient to use. The glass fibre f i l t e r s can be used directly by wetting them briefly with the f i l t r a t i o n buffer, whereas the nitrocellulose f i l t e r s have to be presoaked i n the f i l t r a t i o n buffer for a period of time before use. A high f i l t r a t i o n speed can be used in the f i l t e r binding assays with glass fibre f i l t e r s . Generally, filter-binding assays were performed with f i l t e r speeds of less than 5 ml/min with the nitrocellulose f i l t e r s (36, 57, 77). We have demonstrated that in our filter-binding assay, the retention of PIII-DNA complex is optimal at a f i l t r a t i o n speed of 10-30 ml/min. A speed of 30-40 ml/min has been used with GF/F glass fibre f i l t e r s for assaying the DNA-binding activity of the poly(ADP-ribose) polymerase of of bovine thymus (45). 90 In the cases where the recovery of protein-DNA complex from the f i l t e r i s desired, Coombs et a l . (43) have discussed another advantage of using glass fibre f i l t e r s i n the filter-binding assays. They reported that the elution of the adenovirus DNA-terminal protein complex with sodium dodecyl sulphate from the glass fibre f i l t e r s was 50-fold more efficient compared with the nitrocellulose f i l t e r s . In conclusion, we think the filter-binding assay using GF/C f i l t e r s i s a simple, sensitive and reproducible assay for DNA binding proteins. 2. Mechanism of retention of PIII-DNA complex by the GF/C f i l t e r s We do not understand the mechanism by which the glass fibre f i l t e r s retain the PIII-DNA complex. We are aware that the glass fibre f i l t e r s are commonly used to retain protein or DNA precipitated by trichloroacetic acid or other denaturants. However, the retention of the PIII-DNA complex was clearly not due to DNA precipitation or intermolecular DNA aggregration. This was concluded from the sedimentation analysis of the PIII-DNA complex through the glycerol gradients, since DNA complexed with PHI had a similar sedimentation coefficient as free DNA. We .think that the interaction between the f i l t e r and the protein component of the complex i s most l i k e l y responsible for the retention of the complex. Basic proteins such as albumen, are strongly bound to the surface of the fibres (Glass microfibre f i l t e r s , Whatman Publication No. 824). In fact, GF/C f i l t e r s have been used to retain soluble antigen-antibody complexes through interactions between protein molecules and the f i l t e r s (83). We have not yet determined whether 91 the protein molecules of PHI alone are retained by the GF/C f i l t e r . However, Coombs et a l . (43) have claimed that a l l adenovirus proteins bind to GF/C f i l t e r s . We have also shown that glass fibre f i l t e r s with pore sizes greater than 1.5 ym are less efficient i n retaining the PIII-DNA complex. This result may indicate that the hydrodynamic diameter of the PIII-DNA complex i s less than 1.5 ym. It i s however d i f f i c u l t to envisage that the binding of one or a few molecules of PHI can bring about a drastic change in the hydrodynamic diameter of a PM2 DNA molecule without a shift in the sedimentation coefficient of the DNA. Alternatively, GF/C f i l t e r s with smaller pore sizes might be more efficient because of a higher content of surface materials which interact with the PHI molecules. 3. Comparison of PHI with other UV- or AAAF-DNA-bind ing proteins  from human cel l s PHI i s l i k e l y to be different from an AAAF-DNA-binding protein purified from human placenta by Moranelli and Lieberman (42). The latter protein binds effic i e n t l y to linear duplex T7 DNA treated with AAAF, MMS and MNUA but does not recognise UV-irradiated DNA. PHI, on the other hand, binds ef f i c i e n t l y to supercoiled PM2 DNA treated with UV, AAAF, MNNG but not MMS. PHI i s also different from a UV-DNA-binding protein that has been purified from human placenta by Feldberg and Grossman (40, 41). The latter UV-DNA binding protein binds effic i e n t l y to linear DNA treated with nitrous acid and sodium bisulphite. Whether i t binds to DNA treated with AAAF or MNNG has not been reported. The UV-DNA binding protein elutes from the phosphocellulose column at around 0.175 M potassium phosphate, and has a molecular weight greater than 100,000. PHI, on the other hand, elutes from the phosphocellulose column at around 0.375 M potassium phosphate and has a molecular weight of 20-25,000. There are some similarities between PIII and the UV-DNA unwinding protein from CLL lymphocyte extracts (28). The molecular weight of the unwinding protein is 24,000, which is about the same as PIII. Both proteins are eluted from an UV-irradiated DNA-cellulose column at 1 M NaCl. The binding substrate specificity of the unwinding protein has not been reported. However, the two proteins may di f f e r i n their binding a f f i n i t i e s to single-stranded DNA. The unwinding protein binds tightly to single-stranded DNA-cellulose column and required 2 M NaCl for elution. On the other hand, PIII appears to have a weaker af f i n i t y for single-stranded DNA than UV-DNA. In fact, to our knowledge, the only other naturally occuring protein which binds to DNA treated with UV and AAAF i s the gene 32 protein of phage T4 (cited in reference 84). 4. Biological significance of PIII We infer from the abundance of PIII, which i s probably 10"* molecules per Hela c e l l , that i t must be important in DNA metabolism. Two observations suggest a possible role of PIII in DNA repair. F i r s t , the binding activity of PIII i s damage-dependent. Second, the binding of PIII to UV- or AAAF-DNA has a small dissociation equilibrium constant of 7 x 10-*"1 M which indicates a strong a f f i n i t y of PIII to UV- or AAAF-DNA damage. However, direct evidence for a repair function of PIII i s s t i l l lacking. A DNA-binding protein, which i s probably equivalent to PIII, appears to be present i n the f i b r o b l a s t s of the re p a i r d e f i c i e n t c e l l l i n e s , XP2NE and XP5EG, at si m i l a r l e v e l s as that i n normal human f i b r o b l a s t s . Further, P H I does not possess any s i g n i f i c a n t DNA endonuclease, ^glycosylase, exonuclease or ATPase a c t i v i t y . An argument against a r o l e of P H I i n the excision repair of pyrimidine dimers i s the requirement of high UV-dose f o r the crea t i o n of only a lim i t e d number of binding s i t e s f o r P H I on the DNA. With a UV-dose of 600 J/m2, about 45% of the DNA molecules have a binding s i t e f or P H I while more than 60 thymidine dimers are introduced per DNA molecules (85). There i s also the p o s s i b i l i t y that P H I might a c t u a l l y be a protein involved i n DNA r e p l i c a t i o n or t r a n s c r i p t i o n . UV or other treatments could creat unnatural DNA-binding s i t e s for P H I . Nevertheless, r e c a l l i n g that the uvrA, B and C proteins complement each other to form a UV-endonuclease a c t i v i t y , i t i s possible that P H I may only reveal i t s repair function i n the presence of other proteins. I t i s also i n t e r e s t i n g that P H I binds e f f i c i e n t l y to MNNG-DNA but not MMS-DNA1. MNNG alk y l a t e s the DNA v i a a. S.T1 reaction while N MMS al k y l a t e s the DNA pr i m a r i l y v i a a S N2 reaction (86, 87). MNNG, a N-nitroso a l k y l a t i n g agent, has a higher a f f i n i t y for oxygen i n nuc l e i c acids than MMS (87). One of the alkylated s i t e s on the DNA treated with MNNG i s the O^-position of the guanine residue (86). The 6 0 -alkylguanine residue can base-pair with a thymine residue i n the DNA, and r e s u l t s i n a GC to AT t r a n s i t i o n mutation (88). There i s 1 I r i t h i s discussion, we assume that the protein which binds to AAAF-DNA and UV-DNA also binds to MNNG-DNA and u-DNA. a good correlation between the persistence of 0°-alkylguanine in brain tissue and the frequency of occurence of brain tumors in rats (89, 90). The removal of O^-alkylguanine from the XP group A and XP group C c e l l s was found to be deficient (6, 91). Thus, MNNG may be similar to UV and AAAF in i t s a b i l i t y to induce some DNA lesions which are not repaired e f f i c i e n t l y i n XP ce l l s . In other words, the repair process of certain DNA damage introduced by MNNG, AAAF or UV may be related. Furthermore, pretreatment with' acetylaminofluorene was found to enhance the repair capacity of O^-alkylguanine of the rat l i v e r cells (92). The latter result suggests that the pretreatment may have induced or activated a process or a specific enzyme which i s involved 6 in the repair of 0 -alkylguanine and DNA damage induced by acetylaminofluorene. The dependence of the binding activity of PIII on DNA supercoiling suggests that PIII might unwind the DNA helix. The higher free energy of a supercoiled DNA than an nonsupercoiled DNA favors the binding of proteins which can unwind the DNA double helix and thereby reduce the number of superhelical turns (61). The difference in the equilibrium constants for the binding of an unwinding protein to superhelical and nonsuperhelical DNA can be very large (93). However, we have failed to detect an unwinding effect of PIII with the single-stranded specific endonuclease from Neurospora crassa. It remains to be tested i f PIII w i l l enhance the UV-endonuclease activity of Micrococcus luteus in a similar way as the UV-DNA-unwinding protein of the CLL lymphocytes. 5. Nature of the binding site for PIII A l i k e l y feature of the DNA binding site for PIII i s single-strandness which i s suggested from the a f f i n i t y of PHI to single-stranded DNA and the inhibition of PHI by caffeine. Local denatured regions are known to be present in DNA treated with UV- or AAAF (94-96). Native supercoiled PM2 DNA also contains regions with unpaired bases (at least transiently), particularly i n the A+T-rich regions (97). A bulky nucleotide adduct may enhance the melting of such regions and thereby increase their a f f i n i t y for PHI. Instead of A+T-rich regions, hairpin or cruciform structures (70, 98, 99) may be important for the formation of binding sites for PHI. It has been suggested that the base-pairing in a hairpin structure i s preferentially disrupted by DNA damage (70). But single-strandness alone apparently is not enough for an efficient binding by PHI, since single-stranded DNA i s not bound effic i e n t l y by PHI as compared with the supercoiled DNA damaged with UV, AAAF or MNNG. Thus, DNA supercoiling i s required for an efficient binding of PHI to DNA damage. The PM2 DNA isolated from natural sources i s composed of a Boltzmann distribution of geometrical isomers which differ from each other by an integral number of superhelical turns (100, 101). There are on the average about 90 superhelical turns in the PM2 DNA (102) which has about 9,000-10,000 base-pairs. In eucaryotic c e l l s , the DNA In chromatin i s estimated to have one to two superhelical turns per nucleosome particle (103, 104). The superhelical density may influence the interaction ot proteins with DNA i n two ways. We have already discussed that the free energy associated with the superhelical turns favors the binding to DNA by an DNA-unwinding protein. Alternatively, DNA supercoiling might be necessary for the formation or stabilization of the DNA-recognition site for a protein. The 96 susceptibility of some supercoiled DNA to certain single-stranded specific nucleases i s known to be dependent on the degree of DNA supercoiling (61, 70, 98, 99). For example, PM2 DNA molecules are not sensitive to the Neurospora crassa endonuclease unless they have superhelical densities higher than -0.029 (70). The nuclease-sensitive sites on the supercoiled DNA may be specific and seem to l i e in the loops of potential cruciform structures (99). The cruciform structures may be more stable in DNA with high supercoiled densities and in A+T-rich regions (98, 99). It i s thus possible that the damage-induced binding 6ites of PIII might only be formed on those PM2 DNA molecules with superhelical densities higher than a certain value. Studies of the DNA-binding activity of PIII with DNA of different defined superhelical densities should allow us to determine whether there exists a minimal superhelical density above which the binding sites for PIII are readily induced by DNA damage. There i s another interesting possibility for the nature of the binding sites of PIII. Substitution of a bulky group at the 8-position of the purine nucleotides can cause the purine residues to assume the syn conformation instead of the usual anti conformation (105). For DNA treated with AAAF, the major adduct i s formed by a substitution at the 8-position of the guanine residue (95). UV can also introduce adduct to the 8-position of the purine residues in the DNA. A specific endonuclease activity directed towards the UV-induced 8-alkylated purines i n the PM2 DNA was identified i n extracts of Micrococcus luteus, which probably recognised the syn conformation of the purine residues (105). We plan to determine i f PIII w i l l recognise 97 the UV-induced 8-alkylated purine residues. Interestingly, the syn conformation of the guanine residues i s preferred in the left-handed Z-DNA structure (106). A DNA structure which i s probably assumed by poly(dG-dC)'poly(dG-dC) at salt concentration higher than 2.5 M NaCl (107). By already assuming a syn conformation, the alkylated purine residues favor the structural transition of the DNA from the right-handed B-form to the Z-from at lower salt concentration. Indeed, some residues of poly(dG-dC)•poly(dG-dC) modified with AAAF adopt the Z form even in 1 mM phosphate buffer (108). It i s possible that PIII recognises the syn conformation of purines residues and that the free energy of the binding reaction i s used to induce a structural transition of segments of the DNA from a B-form to a Z-form. 98 Bibliography 1. Lindahl, T. and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochem. 11: 3610-3617. 2. 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