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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.Sc,  M c G i l l U n i v e r s i t y , 1976  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 in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MEDICAL GENETICS  We a c c e p t t h i s t h e s i s a s conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1981  SIU SING TSANG, 1981  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree for  that  understood for  I agree  Library  shall  for reference  and  study.  I  for extensive  that  h i s or  her  copying or  f i n a n c i a l gain  be  shall  publication  not  be  Date  nF-fi  10  /7Q)  2  5  t  h  M a  y>  1 9 8 1  •  the  of  Columbia  make  further this  thesis  head o f  this  It  my  is  thesis  a l l o w e d w i t h o u t my  Medical Genetics  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  by  representatives.  permission.  Department of  copying of  granted  the  University  the  p u r p o s e s may by  the  that  permission  department or  f u l f i l m e n t of  advanced degree at  Columbia,  scholarly  in partial  written  Abstract A DNA-binding protein, P H I , was p a r t i a l l y p u r i f i e d from extracts of Hela c e l l s by high-speed centrifugation, and chromatography on DEAEc e l l u l o s e , 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 p r e f e r e n t i a l l y to supercoiled PM2  treated with u l t r a v i o l e t l i g h t (UV-DNA) or aminofluorene  DNA  N-acetoxy-N-acetyl-2-  (AAAF-DNA) as compared to native supercoiled PM2  DNA.  The binding i s noncooperative. A f i l t e r - b i n d i n g assay u t i l i z i n g GF/C glass f i b r e f i l t e r s used to detect P H I  was  during the p u r i f i c a t i o n steps. Characterisation  of PIII-DNA complex by g l y c e r o l gradient centrifugation indicates that the retention of the complex by the f i l t e r s does not involve DNA p r e c i p i t a t i o n , aggregration, or a conformational change of the  DNA  which r e s u l t s i n a detectable change i n the sedimentation c o e f f i c i e n t of the DNA. PHI  The binding of P H I  to DNA  i s reversible.  i s a protein as indicated by i t s s e n s i t i v i t y to proteinase K.  The sedimentation c o e f f i c i e n t of the protein estimated by g l y c e r o l 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 P H I 100-200 mM NaCl and i s r e l a t i v e l y independent MgCl  2  and MnCl  2  i s optimal at around  of temperature and  at concentrations between 1 mM and 7 mM do not  markedly a f f e c t the binding but i t i s inhibited by sucrose, ATP caffeine.  pH.  and  iii  Competition experiments indicate that P H I  i s a single protein  which binds to AAAF-induced and UV-induced DNA binding s i t e s with equal a f f i n i t y .  PHI  also binds p r e f e r e n t i a l l y to supercoiled PM2  DNA  treated with N-methyl-N'-nitro-nitrosoguanidine but has l i t t l e or no p r e f e r e n t i a l binding a c t i v i t y f o r methyl methanesulphonate-treated depurinated PM2 DNA.  I t also possesses some binding a c t i v i t y to unit  length, single-stranded PM2 DNA.  Nicked or l i n e a r forms of PM2  (damaged or untreated) are not e f f i c i e n t substrates for P H I , a requirement of DNA  or  DNA  indicating  supercoiling for the binding a c t i v i t y of P H I .  possible nature of the DNA-binding s i t e s f o r P H I The b i o l o g i c a l significance of P H I  The  i s discussed.  remains to be determined.  It  does not possess s i g n i f i c a n t glycosylase, endonuclease or exonuclease activities.  The binding of P H I  does not a l t e r the s u s c e p t i b i l i t y of  UV-irradiated supercoiled PM2 DNA to the single-stranded endonuclease of Neurospora crassa.  A DNA-binding protein similar to P H I  was  found  to be present i n extracts of a normal human f i b r o b l a s t c e l l l i n e and two xeroderma pigmentosum f i b r o b l a s t c e l l l i n e s (XP-cell l i n e s ) .  The  concentration of t h i s protein i n the extracts of these c e l l l i n e s was comparable to that of P H I XP5EG and XP2NE.  i n Hela c e l l s .  The two X P - c e l l l i n e s were  They belong to the A and D complementation  of xeroderma pigmentosum, respectively.  groups  The c e l l l i n e XP5EG appeared  to be d e f i c i e n t i n another DNA-binding protein, which eluted from the phosphocellulose column with 180-250 mM potassium  phosphate.  The d i s s o c i a t i o n equilibrium constant f o r the binding reaction of  PHI  t o the UV-  be 7 x 10 ^  M.  or AAAF-induced  b i n d i n g s i t e s on DNA  i s estimated to  The a s s o c i a t i o n r a t e c o n s t a n t and the d i s s o c i a t i o n  r a t e c o n s t a n t a r e 4 x 10^ M *sec * and 3 x 10 ^ s e c \ There a r e a t l e a s t 10"* m o l e c u l e s  of P H I  per H e l a  respectively.  cell.  V  Table of Contents Page Abstract  11  Table of Content  v  L i s t of Tables  viii  L i s t of Figures  xi  Acknowledgement  xii  Abbreviations  xiii  Introduction  1  Materials and Methods  7  1. Tissue culture  7  (a) C e l l l i n e s  7  (b) Culture media  7  (c) Solutions f o r harvesting c e l l s  7  (d) C e l l growth  8  2. Preparation of ^ - l a b e l e d 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. P u r i f i c a t i o n  of the DNA-binding protein, P H I  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 P H I  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 f r a c t i o n s  19  14. S c i n t i l l a t i o n f l u i d  19  15. Miscellaneous  19  Results  21  1. P u r i f i c a t i o n of the DNA-binding protein, P H I  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 P H I 4. Substrate  specificity  33 42  5. Other properties of P H I  51  6. Glycerol gradient sedimentation analysis of P H I  66  7. Characterisation of the PIII-DNA complex  69  8. C a t a l y t i c a c t i v i t y  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 P H I Discussion  85 89  1. Advantages of using glass f i b r e f i l t e r s i n the f i l t e r binding assay  89  vii 2. Mechanism of retention of PIII-DNA complex by the GF/C filters  90  3. Comparison of P H I with other UV- or AAAF-DNA-binding proteins from human c e l l s  91  4. B i o l o g i c a l significance of P H I  92  5. Nature of the binding s i t e f o r P H I  94  Bibliography  98  viii  L i s t of Tables Table  Page  I . P u r i f i c a t i o n o f P H I from H e l a c e l l s II.  Retention  o f PIII-DNA  22  complex by d i f f e r e n t  t y p e s of  Whatman g l a s s m i c r o f i b r e f i l t e r s III.  32  E f f i c i e n c y o f r e t e n t i o n ^ o f PIII-DNA GF/C  complex by the  filters  37  IV. E f f e c t o f DNA c o n f o r m a t i o n on the DNA-binding a c t i v i t y of P H I  52  V. E s t i m a t i o n  o f DNA damage on t h e v a r i o u s  DNA  substrates VI. Substrate  53 s p e c i f i c i t y of P H I  VII. S e n s i t i v i t y of P H I to proteinase  54 K and RNase A  treatment  55  V I I I . E f f e c t o f temperature on the DNA-binding a c t i v i t y o f PHI  59  IX. Freeze-thaw s t a b i l i t y o f P H I  60  X. E f f e c t o f s u c r o s e 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  X I I . Assays f o r UV-DNA endonuclease and g l y c o s y l a s e a c t i v i t i e s o f P H I under v a r i o u s c o n d i t i o n s XIII. Preparation  79  of e x t r a c t s used f o r the a n a l y s e s  of t h e  DNA-binding p r o t e i n s from human f i b r o b l a s t s XIV. Summary o f the a n a l y s e s  o f a UV-DNA-binding  human f i b r o b l a s t e x t r a c t s  82 protein i n 86  lx L i s t of Figures Fig.  Page  1. Chromatography of DNA-binding p r o t e i n s on phosphocellulose  23.  2. UV-DNA c e l l u l o s e chromatography o f t h e p h o s p h o c e l l u l o s e f r a c t i o n of P H I  ..  25.  3. E f f e c t o f N a C l c o n c e n t r a t i o n i n t h e assay m i x t u r e on the DNA-binding a c t i v i t y o f P H I  27.  A. Time course of DNA-binding by P H I 5. R e t e n t i o n  of PIII-DNA  28.  complex on the f i l t e r s as a f u n c t i o n  of t h e NaCl c o n c e n t r a t i o n of t h e d i l u t i o n b u f f e r 6. E f f e c t o f f i l t r a t i o n  29.  speed on t h e r e t e n t i o n o f  PIII-DNA complex  31.  7. DNA-binding o f P H I as a f u n c t i o n o f UV-dose and AAAF-dose  34.  8. DNA-binding as a f u n c t i o n o f t h e amount o f P H I  35.  9. S p e c i f i c b i n d i n g of UV-DNA and AAAF-DNA w i t h v a r i o u s amounts of P H I  39.  10. DNA-binding as a f u n c t i o n of the amount of P H I a t a low low c o n c e n t r a t i o n o f DNA s u b s t r a t e s  40.  11. S p e c i f i c b i n d i n g o f UV-DNA w i t h v a r i o u s amounts o f P H I a t a low c o n c e n t r a t i o n o f DNA s u b s t r a t e s  41.  12. DNA b i n d i n g curve a t low c o n c e n t r a t i o n s of P H I 13.  B i n d i n g o f P H I t o DNA i r r a d i a t e d w i t h h i g h UV-doses  43. ....  44.  14. AAAF-DNA b i n d i n g a c t i v i t y o f P H I i n t h e presence o f competitor  DNA  45.  X  15. A r e c i p r o c a l plot of the data of the competition experiment depicted i n F i g . 14  47  16. Sucrose gradient sedimentation of Msp I-treated DNA 17. E f f e c t of MgCl  2  and MnCl  2  on the binding a c t i v i t y  of P H I 18. DNA  48  57  binding a c t i v i t y of P H I :  pH dependence  58  19. Heat s e n s i t i v i t y of P H I  61  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  63  21. E f f e c t of caffeine on the binding a c t i v i t y of P H I  64  22. Sedimentation v e l o c i t y analyses of P H I  i n the presence  of 0.15 M NaCl  67  23. Sedimentation v e l o c i t y analyses of P H I  i n the presence  of 0.5 M NaCl  68  24. Sedimentation of PIII-UV-DNA complex i n 10-30% g l y c e r o l , and 0 mM NaCl  70  25. Sedimentation of PIII-UV-DNA complex i n 10-30% g l y c e r o l , and 50 mM NaCl  71  26. Sedimentation of PIII-UV-DNA complex i n 10-30% g l y c e r o l , and 150 mM NaCl  72  27. Sedimentation of PIII-u-DNA complex i n 10-30% g l y c e r o l , and 0 mM NaCl  74  28. Sedimentation of PIII-UV-DNA complex formed i n the presence presence of ATP and MgCl  75  2  29. R e v e r s i b i l i t y of the binding of P H I  to UV-DNA  76  xi 30. E f f e c t of P H I  on the s u s c e p t i b i l i t y of DNA to the  single-stranded s p e c i f i c endonuclease from Neurospora  cvassa 31. Phosphocellulose  81 chromatography of DNA-binding proteins  from human f i b r o b l a s t extracts  83  xii Acknowledgement I thank Dr. U. K u h n l e i n  f o r s u p e r v i s i n g my r e s e a r c h .  s t i m u l a t i n g and rewarding t o work w i t h him.  I t has been  I am a l s o g r a t e f u l t o Dr.  H. F. S t i c h f o r h i s c o n s t a n t  support  and  t h e i r time t o be on my s u p e r v i s o r y  Dr. S. Wood f o r d e v o t i n g Mrs.  and Dr. R. M i l l e r , Dr. G. Tener, committee.  G. Wood a s s i s t e d i n some o f t h e t i s s u e c u l t u r e work and Mrs.  J . Koropatnick  prepared  the H-labeled 3  PM2 DNA.  My w i f e , F r a n c o i s e , d e s e r v e s s p e c i a l thanks f o r her p a t i e n c e , encouragement and h e l p . Studentship  awards r e c e i v e d from the N a t i o n a l Cancer I n s t i t u t e of  Canada d u r i n g the p e r i o d of 1978-81 a r e 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) c e l l u l o s e  DMSO  dimethylsulfoxide  DNA  deoxyribonucleic acid  DTT  dithiothreitol  EDTA  disodium ethylene diaminetetraacetate  MMS MMS-DNA MNNG  methylmethanesulphonate PM2 DNA treated with MMS N-methyl-N'-nitro-nitrosoguanidine  MNNG-DNA  FM2 DNA treated with MNNG  MNUA  N-methyl-N-nitrosourea  rpm revolution per min S  sedimentation c o e f f i c i e n t  Tris  tris-(hydroxymethyl)-aminomethane  u-DNA  untreated native PM2 DNA  UV  ultraviolet light  UV-DNA UV-endonuclease  PM2 DNA UV-irradiated 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 f u n c t i o n a l i n t e g r i t y o f 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 spontaneously (2-4).  either  (1) o r by t h e a c t i o n o f c h e m i c a l and p h y s i c a l  agents  I f t h e l e s i o n s a r e n o t c o r r e c t e d by DNA r e p a i r p r o c e s s e s , normal  DNA metabolism  and gene r e g u l a t i o n w i l l be a f f e c t e d .  S e v e r a l t y p e s o f DNA r e p a i r p r o c e s s e s have been proposed and reviewed  (5, 6 ) .  I n f o r m a t i o n c o n c e r n i n g t h e s e p r o c e s s e s has l a r g e l y  been o b t a i n e d from s t u d i e s w i t h p r o c a r y o t e s .  However, human c e l l s  p r o b a b l y a l s o r e p a i r DNA damage by s i m i l a r p r o c e s s e s . S e v e r a l r e p a i r - d e f i c i e n t human g e n e t i c d i s e a s e s have been identified prone  (7, 8).  (9).  I n some o f these d i s e a s e s , t h e p a t i e n t s a r e cancer  Among them, xeroderma pigmentosum i s p r o b a b l y t h e b e s t  characterised  (10, 11).  P a t i e n t s w i t h xeroderma pigmentosum (XP) a r e  v e r y s e n s i t i v e t o s u n l i g h t and a l l o f them have t h e tendency t o develop s k i n tumors. C e l l l i n e s have been e s t a b l i s h e d from t h e s k i n f i b r o b l a s t s o f XP p a t i e n t s .  Except f o r a group  of XP c e l l s c a l l e d XP v a r i a n t , the  f i b r o b l a s t s o f t h e s e X P ' c e l l l i n e s have been shown t o be d e f e c t i v e i n the e x c i s i o n r e p a i r o f UV-induced  thymidine dimers.  I n normal  c e l l s , e x c i s i o n r e p a i r o f the p y r i m i d i n e dimer i s b e l i e v e d t o be i n i t i a t e d by an i n c i s i o n on t h e DNA i n t h e v i c i n i t y o f a dimer. i n c i s i o n i s made e i t h e r by a s p e c i f i c endonuclease endonuclease  a c t i v i t y (UV-  a c t i v i t y ) o r v i a a combination o f a g l y c o s y l a s e  and an a p y r i m i d i n i c endonuclease  a c t i v i t y (12).  activity  The DNA damage and  a d j a c e n t n u c l e o t i d e s a r e then removed by an exonuclease. c r e a t e d i s then f i l l e d w i t h a DNA polymerase  The  The gap thus  a c t i v i t y and f i n a l l y t h e  2  repair patch i s joined to the remaining DNA  by a l i g a s e (5, 6).  The  excision repair deficiency i n the XP c e l l s seems to l i e i n the i n c i s i o n step of the process (10, 11, 13).  Defects of XP.cells i n other.DNA  repair processes have also been reported  (14-17).  C e l l hybridization  studies indicate that the excision repair defect i n XP c e l l l i n e s f a l l s into at least seven complementation groups (10).  This finding suggests  that the i n c i s i o n step of the excision repair pathway for pyrimidine dimers i s a complex process. Furthermore, those XP c e l l s which repair the pyrimidine dimer d e f i c i e n t l y are also defective i n the repair of bulky DNA  lesions  caused by other "UV-like" DNA  damaging agents such as AAAF and  bromobenzanthracene (8, 11).  These XP c e l l s however can repair  p r o f i c i e n t l y DNA MMS  lesions incurred by other damaging agents such as  and X-rays (8, 11).  The DNA  damaging agents of the l a t t e r 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  excision repair of DNA DNA  lesions introduced  damaging agents may  regulatory  (6, 18).  I t i s possible that by UV and by the  the  "UV-like"  share the same repair enzymes or some  proteins.  Other studies have also suggested the existence of  regulatory  molecules i n chromatin which might determine the removal of pyrimidine dimer6 from DNA.  I t was  found that extracts of XP c e l l s from the  complementation groups A and D and the XP variant were capable of excising thymidine dimers from p u r i f i e d UV-irradiated DNA. extracts from c e l l s of the XP group A and  In contrast,  the XP variant did not  excise  dimers from t h e i r endogenous chromatins under conditions where extracts  3  of normal c e l l s and XP group D c e l l s did (19, 20).  However, these  r e s u l t s contradict the repair capacity of i n t a c t c e l l s , where the XP -variant c e l l s but not the XP group D c e l l s exhibit normal excision repair (21).  Nevertheless, i t was  suggested that the XP c e l l s are not  defective i n the UV-endonuclease a c t i v i t y , which must act before the dimers are excised.  Rather, there may  be factors which a f f e c t the  recognition of DNA  damage i n chromatin by the UV-endonuclease, and  some XP c e l l s may  have a deficiency i n one or more of these factors  (19,  20). An added complexity for DNA  the chromatin structure.  repair i n human c e l l s i s imposed by  B a s i c a l l y the chromatin structure i s composed  of repeating units of nucleosome core p a r t i c l e s with the DNA around octamers of histones. the linker-DNA (22). accessible to DNA  DNA  wrapped  These core p a r t i c l e s are connected by  lesions i n the nucleosome core are less  repair enzymes than the l i n k e r DNA  (21, 23-27).  For  human c e l l s i r r a d i a t e d with u l t r a v i o l e t l i g h t , i t has been calculated that the p r o b a b i l i t y of repair synthesis per unit length of DNA  i n the  l i n k e r regions i s 15-fold greater than that i n the core p a r t i c l e s , while there i s no predominance of induction of pyrimidine dimers i n the l i n k e r regions (27).  I t has also been shown that both the  UV-  endonucleases of Micr-ococens tuteus and phage T4 have limited access to the dimer s i t e s i n permeable i r r a d i a t e d human c e l l s (26).  Additional  s i t e s became accessible when the c e l l s were exposed to a high concentration  of NaCl which presumably disrupts the chromatin structure.  Thus, the i n c i s i o n step i n the human excision repair process i s carried out by an endonuclease and by factors which control the a c c e s s i b i l i t y  4  of DNA damage to the putative repair endonuclease. A protein which appears to influence the rate of DNA i n c i s i o n by a UV-endonuclease has been i d e n t i f i e d i n human lymphocytes from patients with chronic lymphocytic leukemia (CLL) (28). This protein was p u r i f i e d by DNA-cellulose chromatography.  I t eluted from a UV-  i r r a d i a t e d c a l f thymus DNA-cellulose column with 1 M NaCl and from a single-stranded DNA-cellulose column with 2 M MaCl. weight of 24,000.  I t had a molecular  This protein can enhance the melting or unwinding  of poly[d(A-T)] and UV-irradiated c a l f thymus DNA but not native c a l f thymus DNA.  Interestingly, the rate of cleavage of UV-irradiated  supercoiled 4>X-174 vj^A by the UV-endonuclease a c t i v i t y of luteus  was enhanced by t h i s unwinding protein.  Micrococcus  Using an immunochemical  procedure, this protein was not detected i n lymphocyte extracts from normal i n d i v i d u a l s .  The presence of t h i s protein might explain  the higher DNA repair capability of CLL c e l l s compared with normal c e l l s (28). We and others have so f a r f a i l e d to purify a pyrimidine dimer s p e c i f i c endonuclease a c t i v i t y from human c e l l s .  These f a i l u r e s may  be due to the small quantity or the l a b i l i t y of the endonuclease a c t i v i t y i n crude extracts of human c e l l s (19, 29).  It i s  possible that the UV-endonuclease a c t i v i t y i s a complex of several protein molecules, which dissociates upon chromatography  leading  to a loss of endonuclease a c t i v i t y . An analogous s i t u a t i o n exists for the UV-endonuclease coded by the uvrA,  B and C genes of Escherichia  coli.  activity  Of the three  UV-endonuclease a c t i v i t i e s p u r i f i e d from procaryotes, the one coded  5  by the uvrA, B and C genes i n Escherichia  coli  i s probably the best  model for the UV-endonuclease a c t i v i t y i n human c e l l s . endonucleases of Micrococcus for  i n XP c e l l s .  DNA  adducts.  UV-  and phage T4 which are s p e c i f i c  pyrimidine dimers, the UV-endonuclease of Escherichia  recognizes bulky DNA  coli  luteus  Unlike the  coli  Such adducts are repaired less e f f i c i e n t l y  Mutations i n the uvrA, B or C genes render  Escherichia  s e n s i t i v e to both UV l i g h t and to agents which can produce bulky adducts (6, 30, 31).  I t has been shown that each of the uvrA,  B or C gene products does not have an appreciable endonuclease a c t i v i t y . They however can complement each other to y i e l d an ATP-dependent endonuclease a c t i v i t y s p e c i f i c for UV-irradiated DNA protein apparently has a molecular weight of 100,000. UV-irradiated superhelical DNA superhelical DNA  (33).  (32).  The  uvrA  It binds to  and to a lesser extent to unirradiated  Recently, a dimer s p e c i f i c endonuclease  a c t i v i t y has been isolated from c a l f thymus (34).  It i s l a b i l e 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 r o l e i n DNA to i s o l a t e proteins which function i n DNA their binding a b i l i t i e s to damaged DNA. incubation of the protein with DNA  repair.  Thus, one approach  repair i s to assay for The simplest assay involves  i n a reaction mixture and  subsequent f i l t r a t i o n of the mixture through a n i t r o c e l l u l o s e f i l t e r . The protein-DNA complex i n the reaction mixture i s retained by the filter.  Such f i l t e r - b i n d i n g assays have been shown to be useful i n  the p u r i f i c a t i o n s of several proteins which are involved or may involved i n DNA  repair.  a c t i v i t y from Micrococcus  be  These proteins included the UV-endonuclease luteus  (35), the T4 endonuclease V  (36),  6 the  UVYA  protein (33), an ATP-independent UV-endonuclease from  c o l i (37),  Escherichia  a DNA-binding protein which can i n s e r t purines into apurinic  s i t e s (38, 39) and the apurinic endonuclease a c t i v i t y from human f i b r o b l a s t s (38).  The f i l t e r - b i n d i n g assays have also allowed the  p u r i f i c a t i o n of two human placental DNA-binding proteins. b i o l o g i c a l functions remain to be determined.  Their  One of them binds to UV-  i r r a d i a t e d DNA but recognizes DNA lesions other than pyrimidine dimers (AO).  I t also binds to DNA treated with nitrous acid or sodium b i s u l f i t e  (Al).  The other protein binds e f f i c e n t l y 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 f i b r e f i l t e r s have also been used i n 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 c e l l s which binds t i g h t l y to c e l l u l a r 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 l a s t protein might have a r o l e i n DNA repair (A6,  A7).  In the hope that we may be able to i s o l a t e an a c t i v i t y which i s involved i n the i n c i s i o n step of excision repair of bulky DNA adducts, we have attempted to p u r i f y DNA-binding proteins from Hela c e l l s which bind p r e f e r e n t i a l l y to UV-DNA and AAAF-DNA.  In t h i s t h e s i s , the p a r t i a l  p u r i f i c a t i o n and characterisation of such a DNA-binding protein i s reported.  We have developed a f i l t e r - b i n d i n g assay using GF/C  f i b r e f i l t e r s f o r the assay of t h i s DNA-binding protein.  glass  In addition,  f i b r o b l a s t s from a normal human c e l l l i n e and c e l l l i n e s of XP group A and XP group D were screened for the presence of t h i s DNA-binding protein.  7  Materials and Methods  1  1. Tissue culture (a) C e l l  lines  Hela c e l l s were purchased from Flow Laboratories, Inc., Rockville, Maryland.  C e l l l i n e 207 was a g i f t from Dr. S. Wood, Department of  Medical Genetics, University of B r i t i s h Columbia.  I t 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 C e l l Repository, I n s t i t u t e of Medical Research, Camden, New Jersey.  C e l l l i n e XP5EG belongs to  the A complementation group of xeroderma pigmentosum and was derived from a skin biopsy of a 23 years old white female.  C e l l l i n e 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 e s s e n t i a l Eagle's medium (Gibco) vas supplemented with 10% f e t a l c a l f serum (Gibco) and the following  antibiotics:  p e n i c i l l i n (100 ug/ml), streptomycin sulphate (30 ug/ml), (100 ug/ml) and fungizone (2.5 ug/ml). from Gibco. bicarbonate.  routinely  kanamycin  The a n t i b i o t i c s were purchased  The medium was adjusted to pH 7.0-7.5 with sodium The culture medium was s t e r i l i z e d by f i l t r a t i o n through a  GSWP M i l l i p o r e membrane f i l t e r with a pore size of 0.2  ym.  (c) Solution f o r harvesting c e l l s Trypsin-EDTA solution was prepared with 8.0 gm of NaCl, 0.2 gm of KH P0 , 0.2 gm of KCl, 1.15 gm of Na HP0 , 0.2 gm of EDTA and 2  4  2  4  0.5 gm of t r y p s i n (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 s t e r i l i z e d by f i l t r a t i o n through  8  a GSWP M i l l i p o r e f i l t e r .  1 l i t e r of phosphate buffered s a l i n e (PBS,  pH 7.1) contained 8 gm of NaCl, 0.2 gm of KC1, 1.15 gm of Na HP0^ and 2  0.2 gm of KB^PO^ and was s t e r i l i z e d by autoclaving. (d) C e l l growth 2 Hela c e l l s were maintained i n 75-cm P l a s t i c s ) with 15 ml of culture media.  tissue culture f l a s k s (Falcon  Four f l a s k s of confluent c e l l s  were pooled and used to inoculate ten r o l l e r bottles (Bellco Biology 2 Glassware).  Each r o l l e r bottle has a surface area of 840 cm .  ml of culture medium was used i n each bottle.  The r o l l e r b o t t l e s were  incubated at 37°C and rotated at a speed of 0.1-0.2 rpm. days, the c e l l s were harvested.  100-150  After 4-6  The c e l l culture medium was decanted,  and the c e l l s were washed b r i e f l y with 10 ml of the trypsin-EDTA solution.  The Hela c e l l s then were detached from the surface of the  b o t t l e s by incubation with another 10 ml of the trypsin-EDTA solution for 5-10 min at room temperature.  During t h i s period the r o l l e r bottles  were rotated at a speed of 3 rpm.  The c e l l s were pelleted by  centrifugation at 200-400 g f o r 6 min.  The p e l l e t was washed three  times with 10 ml of PBS by repeated resuspension and p e l l e t i n g . f i n a l c e l l p e l l e t s were stored i n l i q u i d nitrogen.  The  Ten bottles normally  9 gave 0.5-1.0 x 10 c e l l s . Human f i b r o b l a s t s were grown i n p l a s t i c tissue culture f l a s k s 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 s p l i t 1:3.  2  and 95% a i r .  Confluent c e l l s were  C e l l s were harvested near confluency i n l o t s of 24 f l a s k s 8 with a y i e l d 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.  C e l l s were detached from the tissue culture  f l a s k 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 f o r Hela c e l l s . 2. Preparation of  3 H-labeled PM2  DNA  The PM2 DNA was prepared as described previously (48) except that the x 10  Pseudomonas  g  Bal-31 bacteria were infected at a c e l l density of 3-5  c e l l s / m l with a m u l t i p l i c i t y of i n f e c t i o n of 10 phage per bacterium 3  instead of 2-4 phage per bacterium, and that 2 m C i / l i t e r of methyl- Hthymidine ( s p e c i f i c a c t i v i t y , 25 Ci/mmol, Amersham) was used to l a b e l the PM2 DNA.  The higher m u l t i p l i c i t y of i n f e c t i o n was found to increase  the y i e l d of the PM2 phage. 17-22,000 cpm/yg of  The PM2 DNA  had a s p e c i f i c a c t i v i t y of  DNA. 3  Unlabeled DNA was prepared i n 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 i n 10 mM Tris-HCl, pH 7.5, using a p e t r i dish on i c e and a 60 watt GE G15T8 germicidal lamp.  The incident dose was  measured with a Blak-ray u l t r a v i o l e t meter ( U l t r a v i o l e t 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 f o r 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 d i a l y s e d overnight  t o a n u c l e o t i d e c o n c e n t r a t i o n o f 0.2 mM and  a g a i n s t two changes of 500 m l o f 10 mM T r i s - H C l ,  pH 7.5. Depurination  o f PM2 DNA was c a r r i e d out by h e a t i n g DNA a t a  n u c l e o t i d e c o n c e n t r a t i o n of 0.5 mM a t 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 a t a pH o f 5.0 ( a d j u s t e d w i t h  The  treatment c r e a t e d about 1.5 a p u r i n i c sites/PM2 DNA m o l e c u l e a s  determined by the n i c k i n g assay o f K u h n l e i n MNNG-DNA and MMS-DNA were prepared  e t a l . (49).  by i n c u b a t i n g PM2 DNA a t a  n u c l e o t i d e c o n c e n t r a t i o n o f 1 mM i n 10 mM T r i s - H C l , pH 7.5, concentrations  HCl).  with  various  o f MNNG o r MMS f o r 30 m i n a t 37°C.  S u p e r c o i l e d c i r c u l a r PM2 DNA was converted  t o a l i n e a r form by  i n c u b a t i n g PM2 DNA a t a n u c l e o t i d e c o n c e n t r a t i o n o f 0.1 mM f o r 3 h with  32 u n i t s / m l o f r e s t r i c t i o n endonuclease Msp I (New England  Biolabs)  i n 10 mM T r i s - H C l , pH 7.5, 10 mM M g C ± , 6 mM KC1 and 100 pg/ml o f 2  a c e t y l a t e d BSA. I n experiments where s i n g l e - s t r a n d e d DNA was used, t h e l i n e a r PM2 DNA was e x t r a c t e d f i r s t w i t h an e q u a l volume o f c h l o r o f o r m o c t a n o l (9:1)  and then d i a l y s e d o v e r n i g h t  of 10 mM T r i s - H C l , pH 7.5.  a g a i n s t two changes o f 400 m l  The l i n e a r PM2 DNA was denatured  b e f o r e u s e by a 10-min i n c u b a t i o n i n a b o i l i n g water bath. PM2  DNA was prepared  immediately Nicked  by t r e a t i n g t h e s u p e r c o i l e d c i r c u l a r PM2 DNA w i t h  bovine p a n c r e a t i c DNase I (Sigma).  N a t i v e PM2 DNA a t a n u c l e o t i d e  c o n c e n t r a t i o n o f 0.1 mM was incubated  w i t h 6.5 ug/ml o f DNase I a t 37°C  f o r 1 h i n a r e a c t i o n m i x t u r e c o n t a i n i n g 10 mM T r i s - H C l , pH 7.5, NaCl and 100 ug/ml o f a c e t y l a t e d BSA. a l l the DNA m o l e c u l e s were n i c k e d .  10 mM  A f t e r t h i s treatment v i r t u a l l y  4. DNA-binding assay The standard DNA-binding assay mixture contained 10 mM  Tris-HCl,  pH 7.5, 2 mM EDTA, 139 fmol of H-labeled PM2 DNA molecules (14,0003  18,000 cpm), 175 mM NaCl and an aliquot of protein i n a t o t a l volume of 300 y l i n a b o r o s i l i c a t e test tube.  The mixture was incubated f o r  10 min on i c e . The assay mixture then was diluted with 1.7 ml of i c e cold 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl (buffer G), and f i l t e r e d immediately through a GF/C f i l t e r at a flow rate of 10-30 ml/min. f i l t r a t i o n speed was controlled by a Manostat V a r i s t a l t i c pump.  The The  reaction tube was rinsed once with 1.7 ml of buffer G, and the r e s u l t i n g 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.  Filters  were dried under a heat lamp and the r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n counting.  A unit of DNA-binding a c t i v i t y i s  defined as the amount of protein which r e t a i n s 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. 100 gm of r e s i n was suspended beaker.  Routinely,  i n 2 l i t e r s of d i s t i l l e d water i n a  The r e s i n was allowed to s e t t l e for about 1 h and the  supernatant containing f i n e p a r t i c l e s was decanted.  The procedure was  repeated three times, and the r e s i n was resuspended i n 1 l i t e r of 0.5 M NaOH f o r 20 min. f i l t e r paper.  The suspension was f i l t e r e d through a Whatman No. 1  The r e s i n 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.  I t was s t i r r e d 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  filtered  resuspended  and washed w i t h d i s t i l l e d water. i n 10 mM potassium  F i n a l l y , t h e r e s i n was  phosphate, pH 7.5, and s t o r e d a t 4°C.  (b) U V - i r r a d i a t e d D N A - c e l l u l o s e Cellulose  ( C e l l e x 410, Bio-Rad) was p r e c y c l e d a s d e s c r i b e d by  A l b e r t s and H e r r i c k (50). of  100 gm of c e l l u l o s e was suspended i n 1 l i t e r  e t h a n o l and i n c u b a t e d a t 80°C f o r an hour.  allowed  The c e l l u l o s e was  t o s e t t l e , and t h e e t h a n o l was poured  repeated three times. suspending  off.  The procedure was  The c e l l u l o s e then was s u c c e s s i v e l y washed by  and f i l t e r i n g  a t room temperature  NaOH, 1 mM EDTA and 10 mM H C l .  w i t h 500 m l each of 0.1 M  A f t e r washing w i t h H 0 u n t i l t h e pH 2  of  t h e e f f l u e n t was n e u t r a l , t h e c e l l u l o s e was l y o p h i l i z e d and s t o r e d  at  room  temperature.  A s o l u t i o n c o n t a i n i n g 2 mg/ml o f c a l f thymus DNA i n 10 mM pH  7.4, and 1 mM EDTA ( b u f f e r X) was p r e p a r e d .  i n a p o l y p r o p y l e n e beaker w i t h a diameter for  35 min w i t h a G15T g e r m i c i d a l lamp.  Tris-HCl  40 ml o f t h e DNA s o l u t i o n  of 5 cm was U V - i r r a d i a t e d The i n c i d e n t UV-dose  2 was  12 j/m / s .  stirrer  The DNA s o l u t i o n was mixed v i g o r o u s l y w i t h a magnetic  during the i r r a d i a t i o n .  20 g o f t h e l y o p h i l i z e d c e l l u l o s e was  added t o t h e U V - i r r a d i a t e d DNA s o l u t i o n . out on a g l a s s d i s h w i t h a g l a s s r o d . and a i r d r i e d a t 37°C o v e r n i g h t .  The lumpy m i x t u r e was spread  The d i s h was covered w i t h gauze  A f t e r w a r d s t h e D N A - c e l l u l o s e was  ground t o a powder and l y o p h i l i z e d o v e r n i g h t t o complete t h e d r y i n g procedure.  The d r y D N A - c e l l u l o s e was resuspended  i n 20 ml of 95% e t h a n o l  and U V - i r r a d i a t e d a t a dose r a t e of 10 J/m /s f o r 20 min. 2  c e l l u l o s e was a i r - d r i e d a g a i n a t 37°C o v e r n i g h t . was  resuspended  The DNA-  The D N A - c e l l u l o s e  i n 1 l i t e r o f b u f f e r X and l e f t a t 4°C f o r a day.  then I t was  washed t w i c e by r e s u s p e n s i o n and f i l t r a t i o n w i t h 2 l i t e r s o f b u f f e r X t o  remove f r e e DNA.  F i n a l l y , t h e D N A - c e l l u l o s e was resuspended  i n 100 ml  o f b u f f e r X p l u s 0.15 M NaCl and s t o r e d a s a f r o z e n s l u r r y a t -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 , A l l o p e r a t i o n s were a t 4°C. syringes.  PHI  The columns were made from B-D  plastic  D i a l y s i s was c a r r i e d out w i t h S p e c t r a p o r I d i a l y s i s t u b i n g  w i t h a m o l e c u l a r weight  c u t o f f 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 p o l y p r o p y l e n e o r p o l y e t h y l e n e tubes. (a) Crude e x t r a c t 9 About 2 x 10  H e l a 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 .  H e l a c e l l s were thawed and suspended i n 35 ml o f 50 mM T r i s - H C l , pH 7.5, 1 mM EDTA and 1 mM DTT ( b u f f e r A ) .  The H e l a c e l l s were d i s r u p t e d by  s o n i c a t i o n w i t h s i x 20-sec p u l s e s u s i n g a B i o s o n i k I I I s o n i c a t o r (Bronwill S c i e n t i f i c ) .  The s o n i c a t i o n was performed  s e t t i n g of 30 u s i n g a 4 mm  probe.  a t an i n t e n s i t y  The s o n i c a t e was c e n t r i f u g e d f o r  50 min a t 50,000 rpm i n a Beckman 50 T i r o t o r .  The supernatant  c e n t r i f u g e d once more under i d e n t i c a l c o n d i t i o n s t o i n s u r e removal speed  o f a l l sedimentable m a t e r i a l .  complete  The f i n a l supernatant  supernatant f r a c t i o n ) was r e t a i n e d f o r f u r t h e r  was  (high  purification.  (b) D E A E - c e l l u l o s e chromatography 2 A column (3.8 cm  x 5.3 cm) w i t h 20 ml o f Whatman DE-22 DEAE  c e l l u l o s e was p r e p a r e d and e q u i l i b r a t e d w i t h 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 h i g h speed  supernatant f r a c t i o n was brought  (buffer B).  The  t o the same b u f f e r content  and l o a d e d onto the column a t a f l o w r a t e of 0.25-0.5 ml/min. column then.was washed w i t h b u f f e r B a t the same f l o w r a t e . o f 10 m l were c o l l e c t e d .  The  Fractions  A t o t a l o f 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 w i t h a c t i v i t y were  14  pooled and d i a l y s e d overnight against two changes of 1 l i t e r of 10 mM potassium phosphate, pH 7.4, 10% g l y c e r o l , 1 mM DTT, 1 mM EDTA (buffer C).  The w h i t i s h p r e c i p i t a t e (appearing a f t e r 1-2 h of d i a l y s i s ) was  removed by c e n t r i f u g a t i o n a t 15,000 rpm f o r 15 min i n a Beckman 50 T i rotor.  The supernatant (DEAE-fraction), about 60 ml, was retained f o r  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  of Whatman P - l l phosphocellulose C.  x 8 cm)  p r e v i o u s l y e q u i l i b r a t e d with buffer  The column then was washed w i t h 30 ml of buffer C.  F r a c t i o n s of  10 ml were c o l l e c t e d . The column subsequently was washed w i t h 50 ml of 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 1 mM DTT and 10% g l y c e r o l (buffer D). Afterwards the column was eluted with a 400-ml l i n e a r gradient from 50 mM t o 500 mM potassium phosphate b u f f e r , pH 7.5, containing 1 mM EDTA, 1 mM DTT and 10% g l y c e r o l . Fractions of 6 ml were c o l l e c t e d . The flow rate was 0.3-0.5 ml/min.  The column  f r a c t i o n s were made 40% i n g l y c e r o l and stored at -20°C.  Fractions  containing DNA-binding a c t i v i t y e l u t i n g between 325-425 mM potassium phosphate were pooled (phosphocellulose  f r a c t i o n ) and subjected t o  further chromatography. (d) UV-DNA c e l l u l o s e chromatography 2 A column (0.6 cm  x 5 cm) w i t h 3 ml of UV-irradiated DNA-cellulose  was e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 10% g l y c e r o l , 1 mM EDTA and 1 mM DTT (buffer E ) . A 13-ml a l i q u o t of the phosphocellulose f r a c t i o n was made 100 ug/ml i n B - l a c t o g l o b u l i n and d i a l y s e d against 1 l i t e r of buffer E f o r 3-4 h. The d i a l y s e d e x t r a c t was applied t o the DNAc e l l u l o s e column a t a flow r a t e of about 0.25-0.5 ml/min.  The column  was  washed with 3 ml of buffer E containing 100 yg/ml of B - l a c t o g l o b u l i n  (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.  i n 55% g l y c e r o l and  Fractions were stored  100 ug/ml of B-lactoglobulin at -20°C.  7. Analysis of DNA-binding proteins from human f i b r o b l a s t s The p u r i f i c a t i o n procedures were modified cells.  from those for Hela  B r i e f l y , about 5-6 x 10^ human f i b r o b l a s t s were used i n 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  for 50 min i n a Beckman 50 T i rotor.  centrifuged at 50,000 rpm  The supernatant was  passed  2 through a column (0.64 DEAE-cellulose 0.17 was  ml/min.  cm  x 3.3  cm) of 2 ml of DEAE-cellulose.  The  column then was washed with buffer B at a flow rate of Fractions of 1 ml were c o l l e c t e d .  detected i n the f i r s t 7-8  fractions.  DNA-binding a c t i v i t y  These were combined and  dialysed overnight i n a Spectrapor I d i a l y s i s tubing against changes of 500 ml of buffer C.  The dialysed extract was  two  centrifuged at  15,000 rpm f o r 15 min i n a Beckman 50 T i rotor to remove a p r e c i p i t a t e formed during the d i a l y s i s .  The supernatant (DEAE-fraction) 2  chromatographed on a 3.5-ml phosphocellulose  column (1.1 cm  was 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 c o l l e c t e d .  a 30-ml l i n e a r gradient from 50 mM pH 7.5,  containing 1 mM  of about 0.17  ml/min.  B-lactoglobulin was  The column then was  to 500 mM  EDTA, 1 mM DTT  eluted with  potassium phosphate buffer,  and 10% g l y c e r o l at a flow rate  Fractions of 0.5 ml were collected and  added to a f i n a l concentration of 200 yg/ml.  The  column f r a c t i o n s were assayed immediately for DNA-binding a c t i v i t y .  16  8. Glycerol gradient sedimentation of  PHI  0.2 ml of a diluted aliquot of P H I  was layered on a 4.8 ml, 10-  30% l i n e a r g l y c e r o l gradient i n a polyallomer centrifuge tube.  The  g l y c e r o l 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 b a c i t r a c i n . 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  i n a Beckman SW 50.1 rotor.  Fractions were collected i n polypropylene  tubes from the bottom of the gradient.  BSA (4.25 S), egg white  ovalbumin (3.5 S), a-chymotrypsin (2.5 S), whale s k e l e t a l 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 p r o f i l e 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 under the standard conditions.  reaction was performed  A 200-pl aliquot of the assay mixture  was layered on a 4.8 ml, 10-30% l i n e a r 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. the bottom into polypropylene tubes.  Fractions were collected from  A 50-yl aliquot from each f r a c t i o n  was assayed f o r r a d i o a c t i v i t y .  The remaining portions of the peak  f r a c t i o n s of each gradients were f i l t e r e d through GF/C f i l t e r s as described f o r 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 520% l i n e a r sucrose gradient containing 50 mM Tris-HCl, pH 7.5, and 0.25 M NaCl.  Centrifugation was f o r 3 h at 50,000 rpm and 4°C i n a  Beckman SW 50.1 rotor.  Fractions were collected from the bottom.  A  50-ul aliquot from each f r a c t i o n was assayed f o r r a d i o a c t i v i t y . 11. Enzyme assays Endonuclease a c t i v i t y was assayed by measuring the conversion of supercoiled PM2 DNA to nicked c i r c l e s .  The standard assay f o r DNA  nicking was according to the method of Kuhnlein et a l . (16, 49). 50 y l of the reaction mixture was d i l u t e d with 150 y l of 0.01% of sodium dodecyl sulphate, 2.5 mM EDTA (adjusted to pH 7.0 with HC1). of 0.3 M K HP0 -KOH, pH 12.5, then was added. 2  4  200 y l  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 c i r c u l a r 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 n i t r o c e l l u l o s e membrane f i l t e r paper (Schleicher and Schuell type BA 85, 0.45 ym pore size) which s e l e c t i v e l y retained denatured DNA.  The f i l t e r was washed with 5 ml of 0.3 M NaCl, 0.03 M  sodium c i t r a t e and dried.  The amount of DNA retained on the f i l t e r was.  determined by measuring the r a d i o a c t i v i t y on the f i l t e r by l i q u i d  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 = - l n ( l - X ) (16), where X i s the f r a c t i o n of nicked PM2 DNA molecules. Glycosylase a c t i v i t y was measured by determining the number of a l k a l i - l a b i l e apurinic/apyrimidinic s i t e s introduced into the DNA. assay for a l k a l i - l a b i l e s i t e s was similar to the assay for DNA  The  nicking  except that the 2-min alkali-treatment was replaced by a 1 h incubation with 200 p l of 0.3 M ^HPO^-KOH, pH 12.5, and 50 mM L-lysine at 37°C. This procedure hydrolyses apurinic/apyrimidinic s i t e s (16, 40). Exonuclease a c t i v i t y was measured by determining the amount of radioactive DNA rendered soluble i n 6% t r i c h l o r o a c e t i c acid. For the assay of ATPase a c t i v i t y , aliquots of P H I were incubated i n a reaction mixture containing 139 fmol of PM2 DNA molecules, 1 mM ATP, 2.5 mM M g C l  10 mM Tris-HCl, pH 7.5, and 0.8 uCi/ml of 2, 8- H-ATP 3  2>  (25 Ci/mmol, New England Nuclear). After incubation f o r 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 s t r i p s (0.6 cm x 6 cm) of  polyethyleneimine c e l l u l o s e (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 o r i g i n , and ADP migrates to the middle of the s t r i p .  The s t r i p s were cut into two portions to  determine the amount of radioactive ATP and ADP i n the reaction mixture.  12. Protein determination Protein concentrations were determined by the method of Lowry et al.  (52) or the method of Bradford (53).  was purchased from Bio-Rad Laboratories.  For the l a t t e r assay, the dye BSA (Sigma) was used as  a protein standard i n both methods. 13. Phosphate determination for the column fractions The reagent solution for phosphate determination contained one volume of 10% ascorbic acid and s i x 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. S c i n t i l l a t i o n  fluid  F i l t e r s were counted i n toluene (BDH Chemicals) containing 4 of 2,5-diphenyloxazole (PPO) (Amersham) and 0.1 gm/1  of 1,4-bis[2-(5-  phenyloxyazolyl)]-benzene (POPOP) (Syndel Laboratory). samples ACS s c i n t i l l a t i o n l i q u i d  gm/1  For aqueous  (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 s p e c i f i c endonuclease of Neurospora erassa  (535 units/mg) were purchased from Sigma.  BSA was acetylated with acetic anhydride (Fisher) as described previously (49).  Caffeine, MnCl Sigma.  MgCl-2,  2 >  Tris  ( T r i z m a ) , ATP and ADP were o b t a i n e d from  s u c r o s e and g l y c e r o l were purchased from F i s h e r .  U n l e s s o t h e r w i s e s t a t e d , a l l pH measurements were performed a t room  temperature.  Results 1. P u r i f i c a t i o n of the DNA-binding protein, P H I D e t a i l s of the p u r i f i c a t i o n procedures are described i n Materials and Methods.  The r e s u l t s of a t y p i c a l p u r i f i c a t i o n are summarised i n  Table I. 9  A high speed supernatant f r a c t i o n 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 i n buffer B which contained 0.4 M NaCl.  The DEAE-fraction had a higher DNA-binding  a c t i v i t y than the high speed supernatant f r a c t i o n . r e f l e c t a removal of c e l l u l a r DNA  This r e s u l t might  or another i n h i b i t o r of DNA-binding  a c t i v i t y 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 a c t i v i t y were  separated ( F i g . 1).  The a c t i v i t y which eluted at 325-425 mM  potassium  phosphate bound p r e f e r e n t i a l l y to UV- or AAAF-DNA as compared to the untreated DNA a c t i v i t y was  (u-DNA); with a r a t i o of 6:1.  This peak of DNA-binding  stable for at least a year when stored at r20°C i n the  presence of 40% g l y c e r o l . The two other peaks of DNA-binding a c t i v i t y eluted i n the flowthrough fractions (data not shown) and with 180-250 mM phosphate.  potassium  They did not show any binding s p e c i f i c i t y 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 p r o t e i n ,  designated a r b i t r a r i l y as P H I ,  eluted with 1 M NaCl from the column  22  Table I. P u r i f i c a t i o n of PIII from Hela c e l l s .  Activity, Protein Fraction  I. High speed supernatant I I . DEAE I I I . Phosphocellulose IV. UV-DNAcellulose  Volume  units x 10  Units of a c t i v i t y x 10 per mg of protein  mg  ml  UV-DNA  u-DNA  UV-DNA  u-DNA  350  40  540  250  1.6  0.7  300  60  750  400  2.5  1.3  2.6  117  270  45  104  17  _2  54  95  14  -  -  1  3  Volume of the f r a c t i o n i n 40% g l y c e r o l . •The amount of protein was not determined. 'Volume of the f r a c t i o n i n 55% g l y c e r o l .  23  Fraction F i g . 1.  Chromatography of DNA-binding p r o t e i n s on p h o s p h o c e l l u l o s e . Assays were performed w i t h UV-DNA ( • ) , AAAF-DNA ( • ) or u-DNA ( O ) as the b i n d i n g s u b s t r a t e s . Phosphate concentration ( ) .  (Fig. 2).  This p u r i f i c a t i o n step separated  PHI  from another  binding a c t i v i t y which eluted from the column with 0.5 M NaCl. f r a c t i o n s of P H I  DNAPeak  were stored i n 55% g l y c e r o l and 100 ug/ml  6-lactoglobulin with no l o s s i n DNA-binding a c t i v i t y for at least half a year.  Routinely, 100 ug/ml of 6-lactoglobulin was  included i n the  e l u t i o n buffers for the DNA-cellulose chromatography of  PHI.  It has been reported that c a r r i e r proteins such as lysozyme a f f e c t the e l u t i o n of steroid receptors on DNA-cellulose chromatography (55). We have not observed any e f f e c t of 6-lactoglobulin on the e l u t i o n of PHI  from the UV-irradiated DNA-cellulose column.  p r o f i l e as that shown i n F i g . 2 was was  A similar elution  obtained when the chromatography  c a r r i e d out with e l u t i o n buffers without B-lactoglobulin.  i f stored i n the absence of 6-lactoglobulin, P H I of i t s a c t i v i t y i n 16 h at 4°C. storage of P H I  However,  l o s t at least half  Other attempts which included  the  i n the absence of c a r r i e r protein at -20°C or i n l i q u i d  nitrogen with and without 55% g l y c e r o l f a i l e d to s t a b i l i z e BSA or acetylated BSA  can be used to s t a b i l i z e P H I ,  PHI.  but we have  chosen a r b i t r a r i l y 6-lactoglobulin. The p u r i f i c a t i o n of P H I at l e a s t 40-fold.  a f t e r phosphocellulose  chromatography was  The r a t i o of AAAF- or UV-DNA-binding a c t i v i t y  r e l a t i v e to u-DNA-binding a c t i v i t y increased from about 2:1 i n the f i r s t two f r a c t i o n s to about 7:1 i n the f i n a l f r a c t i o n .  This indicates  an enrichment of the s p e c i f i c binding a c t i v i t y for AAAF-DNA and UV-DNA during the p u r i f i c a t i o n .  0.15 M NaCl  - i  0.5 M NaCl  *  1.0 M NaCl  *  2.0 M NaCl  *  Fraction F i g . 2.  UV-DNA c e l l u l o s e chromatography o f t h e p h o s p h o c e l l u l o s e fraction of P H I . Assays were performed w i t h UV-DNA ( • ) , AAAF-DNA (•) or u-DNA ( O ) as t h e b i n d i n g s u b s t r a t e s .  26 2. Properties of the DNA-binding assay Basically,  the DNA binding assay consisted of four steps: (1)  incubation of P H I with DNA; (2) d i l u t i o n of the assay mixture; (3) filtration  of the mixture through GF/C glass f i b r e 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 The standard  buffer.  conditions f o r the DNA-binding assay were established  by studying several parameters.  F i r s t , the influence of NaCl  concentration on the DNA-binding a c t i v i t y of P H I was investigated. Fig. 3 indicates that the binding of P H I to UV- or AAAF-DNA was optimal at 100-200 mM NaCl.  Thus, P H I can bind optimally to UV-  or AAAF-DNA at i o n i c strength near physiological conditions.  A salt  concentration of 175 mM NaCl was used i n the standard assay mixture. The binding of P H I to DNA was very fast and an incubation of two min at 0°C was s u f f i c i e n t for the establishment (Fig. 4). Therefore, our standard  of an equilibrium  incubation condition of 10 min  was more than adequate. The next condition we studied was the NaCl concentration of the d i l u t i o n buffer used i n the second step of the DNA-binding assay. I t should be noted that the binding reaction could s t i l l occur i n this second step.  The r e s u l t depicted i n F i g . 5 indicated that the amount  of UV-, AAAF- or u-DNA retained on the f i l t e r by P H I was maximal at 20-50 mM NaCl.  Since we were interested primarily i n the s p e c i f i c  binding of P H I to UV-DNA and AAAF-DNA, a d i l u t i o n buffer with 0.1 M NaCl was chosen to minimize the nonspecific binding of P H I to DNA and/or the retention by the f i l t e r s of complex formed v i a the nonspecific binding of P H I 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  100  200 N a C l  F i g . 3.  ,  m  300  M  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 . 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 d i l u t e d 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 a c t i v i t y was measured with UV-DNA (• ) , AAAF-DNA ( • ) or u-DNA ( O ) .  28  F i g . 4.  Time course o f DNA-binding by P H I . DNA-binding assays were performed under t h e standard c o n d i t i o n s f o r v a r i o u s i n c u b a t i o n times w i t h UV-DNA ( • ) , AAAF-DNA ( • ) o r u-DNA ( O ) a s t h e b i n d i n g substrates.  29  50  150 N a C l ,  Fig.  5.  250 m M  R e t e n t i o n o f PIII-DNA complex by the f i l t e r s as a f u n c t i o n o f t h e N a C l c o n c e n t r a t i o n o f the d i l u t i o n buffer. The DNA-binding a s s a y s were performed under t h e s t a n d a r d c o n d i t i o n s except t h a t t h e NaCl c o n c e n t r a t i o n i n t h e d i l u t i o n b u f f e r was v a r i e d as i n d i c a t e d . DNA-binding a c t i v i t y was a s s a y e d w i t h UV-DNA ( • ) , AAAF-DNA (• ) or u-DNA ( O ) .  protein complex with an equal e f f i c i e n c y at various NaCl  concentrations  from 0.15 M to A M NaCl (A3, AA). A similar phenomenon has been observed i n the binding of bovine thymus poly(ADP-ribose) polymerase to nicked DNA.  A s a l t concentration of 0.1 M NaCl also was used  to i n h i b i t 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 i n a substantial loss of the amount of PIII-DNA complex retained on the f i l t e r s ( F i g . 6 ) . a flow rate of 10-30 ml/min.  Thus we usually f i l t e r e d at  I t i s well documented that a slow flow  rate r e s u l t s i n a more reproducible and greater retention of proteinDNA complex by n i t r o c e l l u l o s e f i l t e r s (56, 5 7 ) . loss of PIII-DNA complex with low f i l t r a t i o n  The reason for the  speeds remains to be  determined. There are f i v e d i f f e r e n t kinds of Whatman GF grade glass f i b r e f i l t e r s which d i f f e r i n their thicknesses and pore sizes (Table I I ) . The pore size of each f i l t e r i s defined as the size of the p a r t i c l e s that can be retained by the f i l t e r with an e f f i c i e n c y 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 a b i l i t y to r e t a i n PIII-DNA complex.  F i l t e r s with  pore sizes less than 1.5 ym retained the PIII-DNA complex e f f i c i e n t l y (Table I I ) . Usually, assays were performed i n 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 i n duplicate, the duplicates usually agreed to within 10%. In the absence of P H I , the amount of DNA retained on the f i l t e r s  31  Fig.  6.  E f f e c t of the f i l t r a t i o n speed on the r e t e n t i o n of P I I I DNA complex. The b i n d i n g - a s s a y s were performed w i t h UV-DNA ( • ) or u-DNA ( O ) a t the v a r i o u s f i l t r a t i o n speeds i n d i c a t e d . Each p o i n t s i s the average of d u p l i c a t e a s s a y s . The f i l t r a t i o n time was t h e time needed f o r a 2-ml a s s a y m i x t u r e t o pass through a f i l t e r .  32  Table I I .  Retention of P I I I - D N A complex by d i f f e r e n t 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 s i z e , ym  UV-DNA  u-DNA  GF/A  0.25  1.6  11.5  1.9  GF/B  0.71  1.0  13.2  4.0  GF/C  0.25  1.2  12.0  2.9  GF/D  0.65  2.7  8.5  2.8  GF/F  0.44  0.7  12.2  2.4  The DNA-binding assays were performed under the standard conditions with d i f f e r e n t types of Whatman glass f i b r e f i l t e r s . The data were the averages of duplicate assays.  33  (background) was we DNA  have used. was  l e s s than 1.5%  f o r the v a r i o u s k i n d s o f duplex PM2  The background f o r the u n i t l e n g t h s i n g l e - s t r a n d e d  even lower and was  0.3-0.5% of the i n p u t DNA.  i n t h e p r e s e n c e of  PM2  Where a p p r o p r i a t e ,  the backgrounds were s u b s t r a c t e d from the amount of DNA the f i l t e r s  DNA  r e t a i n e d on  PHI.  3. Formation o f PIII-DNA complex as a f u n c t i o n o f the amount of DNA  damage and An  the c o n c e n t r a t i o n of  a l i q u o t of P H I  which c o n t a i n e d  a c t i v i t y o n l y r e t a i n e d 2.4  amount of PIII-DNA complex was J/m  u n i t s of UV-DNA-binding  2  The  amount  the f i l t e r i n c r e a s e d when the  t r e a t e d w i t h i n c r e a s i n g dose of UV  AAAF-dose reached 1,200  23.5  fmol of u-DNA on the f i l t e r .  of PIII-DNA complex r e t a i n e d on was  PHI  or AAAF ( F i g . 7 ) .  DNA  The maximum  r e t a i n e d when the UV-dose and  and 0.01  mg/ml, r e s p e c t i v e l y .  the The  result  2 a l s o suggested t h a t DNA  UV-irradiated with  equivalent  to DNA  for  Thus, UV-DNA and AAAF-DNA were prepared  PHI.  treated with  0.01  a dose o f 1,200  J/m  mg/ml AAAF as a b i n d i n g  used i n experiments where the b i n d i n g of P H I  substrate  accordingly  to both DNA  was  and  substrates  were compared. Under the standard  c o n d i t i o n s , the r e t e n t i o n of DNA  dependent on the amount of P H I concentrations complex was  of P H I ,  (Fig. 8).  However, a t  and AAAF-DNA s t a r t e d t o l e v e l o f f .  higher  the b i n d i n g curves We  have e s t i m a t e d  DNA-binding a c t i v i t y of the v a r i o u s a l i q u o t s of P H I  The  linearly  t h a t i s , when more than 40 fmol of PIII-DNA  r e t a i n e d by the f i l t e r s ,  p o r t i o n of t h e b i n d i n g  was  f o r UV-DNA  the u n i t s of from the  linear  curves.  l e v e l i n g o f f of the b i n d i n g curves might be due  l i m i t e d b i n d i n g c a p a c i t y of the f i l t e r s ;  to (1) a  (2) a low r e t e n t i o n e f f i c i e n c y  34  30  20  /  0  E L Q) •P  10  F  C  UV  TJ d) C (D •P Q) L  <  2 •  ±  1000  0  ±  dose,  J/rr.2  30  20  10  V 0.01 A A A F  F i g . 7.  ±  2000  0.02 dose,  mg/ml  DNA-binding of P H I as a function of UV-dose and AAAF-dose. The DNA-binding assays were performed under the standard conditions with an aliquot of P H I containing 23.5 units of UV-DNA-binding activity.  20  40  60  Plll.pl F i g . 8.  DNA-binding as a function of the amount of P H I . The DNA-binding assays were performed under the standard conditions with various amounts of P H I . P H I had a UV-DNA-binding a c t i v i t y of 2.5 u n i t s / y l . 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 s i t e s . In order to r u l e out p o s s i b i l i t y (1), two reaction mixtures containing P H I  with 100 units of UV-DNA-binding a c t i v i t y were f i l t e r e d  through the same glass f i b r e f i l t e r . about 140 fmol of DNA,  which was  single reaction mixture was  The amount of DNA  retained  was  twice the amount retained when a  filtered  (Fig. 8).  capacity of the f i l t e r was not l i m i t i n g .  Thus the binding  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 r e s u l t i n a s i g n i f i c a n t e l u t i o n of the PIII-DNA complex retained during the f i r s t  filtration.  The retention e f f i c i e n c y of the glass f i b r e 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  and was not present i n the f i l t r a t e which passed through the filter  filter,  first  (Table I I I ) . Assuming that i n t e r a c t i o n with the f i l t e r does not  cause the d i s s o c i a t i o n of the PIII-DNA complex, the retention e f f i c i e n c y of the f i l t e r i s close to  100%.  The formation of complex between P H I was  l a r g e l y due to the binding of P H I  on the DNA  ( s p e c i f i c binding of P H I  with a saturating amount of P H I ,  and UV-  to UVto UV-  or AAAF-DNA  or AAAF-induced s i t e s  or AAAF-DNA) .  a s i g n i f i c a n t amount of complex i s  expected to be formed as a r e s u l t of the binding of P H I are not induced by DNA binding to UV-  damage.  However  to s i t e s that  To estimate the amount of s p e c i f i c  or AAAF-DNA for a saturating amount of P H I ,  points i n F i g . 8 were each corrected by using the  equation:  the data  Table I I I . E f f i c i e n c y 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 Experiment I  II  no. of filter  u--DNA  +PIII  -PHI  +PIII  -PHI  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  1st  30.8  1.3  4.4  0.7  In experiment I, each DNA-binding assay mixture was f i l t e r e d through three f i l t e r s stacked on top of each other. In experiment I I , each assay mixture was f i l t e r e d through one f i l t e r . The data are averages of duplicate assays.  38  y =  A  /  - x 100% 100% - B/0.8 0  8  B / 0  (1)  8  where y i s the % s p e c i f i c 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 i n a l a t e r section, P H I d i d 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 F i g . 9. A plateau l e v e l of DNA retention i s approached when the amount of P H I i n the assay mixtures exceeds 100 units of UV-DNA-binding a c t i v i t y .  The  plateau corresponds to a retention of about 55% of the supercoiled UV- or AAAF-DNA.  I t should be noted that equation (1) i s only v a l i d  for a saturating amount of P H I . For a nonsaturating i t leads to an overestimation  amount of P H I ,  of the amount of s p e c i f i c binding of  P H I to UV- or AAAF-DNA since the binding a f f i n i t y of P H I to UV- or AAAF-DNA i s about 30-fold higher than u-DNA (see l a t e r section e n t i t l e d "Substrate s p e c i f i c i t y " ) . F i g . 10 shows the binding curves f o r P H I determined at a DNA concentration 16-fold lower than that used i n the standard conditions.  assay  The DNA was UV-irradiated with a dose of 1,200 J/m or  600 J/m . Again, the data points i n F i g . 10 are corrected by using equation (1) to plot the s p e c i f i c binding curves shown i n F i g . 11. With saturating amounts of P H I , the s p e c i f i c binding curve for DNA UV2 i r r a d i a t e d 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 b i n d i n g o f UV-DNA and AAAF-DNA w i t h v a r i o u s amounts of P H I . The d a t a p o i n t s f o r UV-DNA ( • ) or AAAF-DNA ( • ) were c a l c u l a t e d from the r e s u l t s of F i g . 8 as d e s c r i b e d i n text.  40  Protein,  F i g . 10.  J_I I  DNA-binding as a function of the amount of P H I 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 P H I as indicated. The UV-DNA-binding a c t i v i t y of P H I used was 3.3 u n i t s / u l . DNA-binding a c t i v i t y was assayed with PM2 DNA UV-irradiated at 1200 J/m ( # ) , PM2 DNA UV-irradiated at 600 J/m ( A ) and u-DNA ( O ) . Each point is.the average of duplicate assays. 2  2  41  PI I  F i g . 11.  I , Jjl  Specific binding of UV-DNA with various amounts of P H I at a low concentration of DNA substrates. The data p o i n t s f o r DNA UV-irradiated with 1,200 J/m (• ) or 600 J/m ( A ) were calculated from the r e s u l t s shown i n F i g . 10 as described i n text. 2  42 DNA.  For DNA  UV-irradiated with a lower dose of 600 J/m  2  , a lower  plateau value which corresponds to 45% of the supercoiled PM2 obtained.  These r e s u l t s can be interpreted as follows. 2  i r r a d i a t i o n of DNA  at doses of 1,200  and 45% of the supercoiled DNA s i t e s for P H I ,  J/m  DNA i s  After  2 and 600 J/m  , about 65%  contained at least one UV-induced binding  respectively. This i n t e r p r e t a t i o n i s only v a l i d i f every  PIII-DNA complex formed i n the assay mixture was retained by the f i l t e r and i f the binding of one P H I retention of a PM2  molecule was  DNA molecule.  enough to cause the  The f i r s t assumption was  discussed i n the previous paragraphs.  already  The second assumption seems to  be v a l i d since DNA-binding i s l i n e a r at low concentration of where DNA  i s i n excess (Fig. 12).  If the retention of DNA  PHI,  required more  than one binding event, a sigmoidal binding curve would be expected (56). The l i n e a r i t y of the binding curve also indicated that the binding of PHI  to DNA i s noncooperative. With higher UV-doses more P H I  be introduced. DNA (Fig.  With a UV-dose of 3,600 J/m  (or 90% of the supercoiled DNA) 13).  was retained on the  filters  was  l i k e l y due to limited binding s i t e s on the UV-  or  molecules.  4. Substrate Fig.  , about 70% of the t o t a l  Thus, the l e v e l i n g off of the binding curves depicted  i n F i g . 8-11 AAAF-DNA  binding s i t e s per DNA molecule can 2  specificity  14 i l l u s t r a t e s 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 P H I was nearly eliminated.  This suggested that P H I  to AAAF-DNA  i s a single protein  43  F i g . 12. DNA-binding curve at low concentration of P H I . The DNA-binding assays were performed under the standard conditions with various amounts of P H I . P H I used has a UV-DNA-binding a c t i v i t y of 2.5 u n i t s / u l . The binding substrates were UV-DNA (•) or u-DNA ( O ) . Each point i s the average of duplicate assays.  44  80 L  (1)  C 0  60  TJ  QJ  £  40  CD  /  Q) L  <  z •  20  UV F i g . 13.  dose,  J/m  2  x  10"  3  B i n d i n g o f P H I t o DNA i r r a d i a t e d w i t h h i g h UV-doses . The DNA-binding a s s a y s were performed under the standard c o n d i t i o n s w i t h 8.7 f m o l of U V - i r r a d i a t e d PM2 DNA. Each assay was c a r r i e d out w i t h an a l 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  |  4  Ratio  I  I  I  I  1  L_  8 12 16 20 24 28 of  competitor  D N A to A A A F D N A  F i g . 14. AAAF-DNA-binding a c t i v i t y of P H I i n the presence of competitor DNA. H-AAAF-DNA binding a c t i v i t y of P H I was assayed under the standard conditions i n the presence of various amounts of unlabeled UV-DNA (•) or u-DNA (O). 3  32  which binds to both UV-DNA and AAAF-DNA. P H I 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 a c t i v i t y by 50%.  A r e c i p r o c a l plot of the data of the competition  experiment was made according to Spillman et a l . (58) to determine the r e l a t i v e binding a f f i n i t y of P H I to the d i f f e r e n t DNA substrates (Fig. 15).  With UV-DNA as the competitor, the r e c i p r o c a l plot y i e l d s  a straight l i n e with a slope close to 1, indicating that P H I 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 r e c i p r o c a l plot y i e l d s a straight l i n e with a slope of about 0.03.  Thus, P H I 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  s i t e s per DNA molecule on the AAAF-DNA and UV-DNA are about equal (Fig. 9), we can conclude that P H I has the same a f f i n i t y towards the AAAF-induced and UV-induced DNA-binding s i t e s . To investigate the effect of DNA conformation on the binding a c t i v i t y of P H I , supercoiled AAAF-DNA, UV-DNA and u-DNA were nicked with bovine pancreatic DNase I or converted to a l i n e a r form by treatment with the r e s t r i c t i o n endonuclease Msp I. isoschizomer of Hpa I I . CCGG.  Msp I i s an  Both r e s t r i c t i o n enzymes recognize the sequence  However, i n contrast to Hpa I I , Msp I cleaves the DNA even i f  the i n t e r n a l cytosine i s methylated  (59, 60).  double-stranded cut per PM2 DNA molecule (61).  Hpa I I makes only one Sucrose gradient  analyses of PM2 DNA cleaved by Msp I indicated that most of the c i r c u l a r form of PM2 DNA was converted to the l i n e a r form ( F i g . 16). The l i n e a r form of PM2 DNA sedimented c i r c u l a r form of PM2 DNA.  s l i g h t l y slower than the nicked  This i s analogous to the observation that the  l i n e a r HF I I I form of <j>X174 phage DNA has a smaller S value than the  47  /  1  4  1  8  I  I  I  I  I  L_  12 16 20 24 28 32  Ratio of competitor  D N At o A A A F D N A  F i g . 15. A r e c i p r o c a l plot of the data of the competition experiment depicted i n F i g . 14. x i s the r a t i o of the amount of AAAF-DNA retained on a f i l t e r i n the presence of competitor DNA to the amount of AAAF-DNA retained i n 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 i n Materials and Methods. (A) DNA which was UV-irradiated with 1200 J/m ( • ,O). (B) DNA which was treated with 0.02 mg/ml of AAAF ( • , • ) . (C) untreated DNA ( A , A). The sedimentation was from r i g h t to l e f t . 2  B  F i g . 16 (B).  c  F i g . 16 (C).  nicked c i r c u l a r RF I I form (62). It i s clear from Table IV that P H I prefers supercoiled DNA as the binding substrate.  P H I binds to the l i n e a r or relaxed c i r c u l a r  form of UV- or AAAF-DNA 5- to 10-fold l e s s e f f i c i e n t l y than to the supercoiled form of DNA. We have also tested the binding a c t i v i t y of P H I towards DNA treated with two a l k y l a t i n g agents, MNNG and MMS.  Using d i f f e r e n t  types of f i l t e r - b i n d i n g assays f o r DNA lesions, the extent of DNA a l k y l a t i o n can be estimated ( 4 9 ) . The l e v e l s of a l k y l a t i o n i n the various MMS-DNA preparations were similar to or greater than the two MNNG-DNA preparations (Table V) .  The binding a c t i v i t y of P H I assayed  with DNA treated with 1 mM MNNG was about 40% of that assayed with UVor AAAF-DNA.  DNA treated with 5 mM MNNG was at least as e f f i c i e n t a  substrate f o r P H I 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 s i t e s for P H I (Table VI) . PHI  did not bind e f f i c i e n t l y to depurinated DNA with about 1.5  apurinic s i t e s .  The binding a c t i v i t y with single-stranded DNA as  a substrate was the same as with u-DNA.  The l a t t e r r e s u l t suggests  that the single-strandness of the DNA alone does not account f o r the DNA-binding a c t i v i t y of P H I . 5. Other properties of P H I DNA-binding a c t i v i t y of P H I 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 P H I was not i n h i b i t e d , i n d i c a t i n g that proteinase K did not interfere with the formation of PIII-DNA complex (Experiment I and I I , Table V I I ) . We therefore concluded that the DNA-binding a c t i v i t y of P H I was due  Table IV.  E f f e c t of DNA conformation on the DNA-binding a c t i v i t y of F i l l .  DNA retained on f i l t e r , fmol Treatment  AAAF-DNA  -Msp I  15.4 (100%)  +Msp I  3.2 (21%)  UV-DNA  u-DNA  15.4 (100%)  1.4 (9%)  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 i n Materials and Methods. The values i n parentheses were % DNA-binding a c t i v i t y r e l a t i v e to that assayed with supercoiled UV-DNA.  53 Table V. Estimation of DNA damage on various DNA substrates.  Average number of nicks (or f i l t e r - b i n d i n g s i t e s ) per DNA molecule DNA  Assay I  Assay II  Assay I I I  Assay IV  MNNG-DNA (5 mM)  0.74  1.58  0.88  3.10  (1 mM)  0.40  0.67  0.43  • 1.12  1.23  >3.50  1.67  >3.50  MMS-DNA (100 mM) (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 i s the standard nicking assay as described i n Materials and Methods. I t measures mainly s i n g l e - and double-stranded breaks i n DNA and to a certain extent l o c a l distortions of DNA. Assay I I i s the standard nicking assay except that the DNA i s 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 I I , respectively, except that the DNA was incubated at 70 C f o r 7.5 min before the nicking assays were performed. The amount of a l k a l i - l a b i l e s i t e s i n the DNA can be calculated by subtracting the results of assay I from those of assay I I . The amount of heat-induced a l k a l i - l a b i l e s i t e s can be estimated by subtracting the results of assay I I I and the amount of a l k a l i - l a b i l e s i t e s from those of assay IV. The amount of DNA breakage detected by the various assays generally increases with increasing l e v e l of DNA a l k y l a t i o n . For a discussion of the scope of the various types of DNA damage that can be measured with these assays and the l i m i t a t i o n s of the assays, see Kuhnlein et a l . (49).  Table VI.  Substrate  Substrate  s p e c i f i c i t y of P H I .  % activity  UV-DNA  100  AAAF-DNA  100  u-DNA  12  MNNG—DNA (5 mM)  97  (1 mM)  39  (100 mM)  10  (40.mM)  15  (20 mM)  12  (10 mM)  10  MMS-DNA  depurinated  DNA  s i n g l e - s t r a n d e d DNA  18 10  DNA-binding a c t i v i t i e s were assayed under t h e standard c o n d i t i o n s w i t h t h e v a r i o u s DNA s u b s t r a t e s . 15-20 u n i t s (UV-DNA-binding a c t i v i t y ) o f P H I were used i n these experiments. The d a t a f o r UV-DNA, d e p u r i n a t e d DNA and s i n g l e - s t r a n d e d DNA a r e t h e averages of d u p l i c a t e assays. The v a r i o u s DNA s u b s t r a t e s were prepared as d e s c r i b e d i n M a t e r i a l s and Methods. The b i n d i n g a c t i v i t y towards s i n g l e - s t r a n d e d DNA has been m u l t i p l i e d by two s i n c e b i n d i n g t o one double-stranded DNA m o l e c u l e g i v e s twice t h e amount o f r a d i o a c t i v i t y a s t h e b i n d i n g o f one s i n g l e - s t r a n d e d DNA m o l e c u l e .  55  Table VII.  S e n s i t i v i t y of P H I to proteinase K and RNase A treatments  Incubation at Experiment I  II  III  DNA retained on f i l t e r , fmol  Treatment  37°C, min  UV-DNA  u-DNA  -Proteinase K  30  14.2  2.8  +Proteinase K  30  0.3  1.0  -Proteinase K  0  20.8  4.0  +Proteinase K  0  20.1  3.5  -RNase A  30  7.6  1.1  +RNase A  30  7.3  0.8  Proteinase K solutions (1 mg/ml) were prepared i n 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 P H I were incubated with 20 ug/ml of proteinase K or 100 ug/ml of RNase A at 37 C f o r various periods of time as indicated. The treated aliquots of P H I were then assayed f o r DNA binding a c t i v i t i e s under the standard conditions.  to protein molecules.  In addition, RNA appears not to be involved i n  the DNA-binding by P H I ,  since P H I i s not sensitive to RNase A  (Experiment I I I , Table VII). The presence of 1-7 mM MgC^ or MnC^ i n the assay mixtures did not markedly a f f e c t the binding of P H I to UV-DNA and u-DNA. (Fig. 17). The binding a c t i v i t y of P H I was also measured with the assay mixture buffered with either Tris-HCl or potassium phosphate at d i f f e r e n t pH values.  P H I bound to UV- or u-DNA to a similar extent  at pH values between 6 and 9 ( F i g . 18).  V a r i a t i o n of the incubation  temperature of the reaction mixture between 0°C and 37°C d i d not a f f e c t the DNA-binding a c t i v i t y of P H I s i g n i f i c a n t l y (Table VIII). The DNA-binding a c t i v i t y of P H I Is quite r e s i s t a n t to freeze-thaw treatment.  In two separate experiments, less than 25% of the UV-DNA-  binding a c t i v i t y of P H I was l o s t by freezing and thawing several times (Table IX).  P H I i s also quite heat stable.  When incubated for  50 min at 60°C i n 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, P H I l o s t only 30-40% of i t s binding a c t i v i t y to UV-DNA (Fig. 19).  The i n a c t i v a t i o n of the binding a c t i v i t y of P H I to u-DNA  was f a s t e r , a loss of 70-80% of the binding a c t i v i t y was observed i n 50 min at 6Q°C.  This r e s u l t suggests that the UV- or AAAF-DNA-binding  a c t i v i t y of P H I may be contaminated with a protein (or proteins) which binds only to u-DNA.  The l a t t e r may be p u r i f i e d from the former by  g l y c e r o l gradient sedimentation (see l a t e r section and F i g . 23). Three chemicals were found to i n h i b i t the DNA-binding a c t i v i t y  57  F i g . 17. Effect of MgCl and MnCl- on the binding a c t i v i t y of PHI. The DNA-binding assays were performed under the standard conditions i n the presence of various concentrations of MgCl ( # , 0 ) or MnCl2 ( A , A ) i n the assay mixtures. DNA-binding a c t i v i t y was assayed with UV-DNA ( • , A ) or u-DNA ( O , A ) . ?  2  58  30h  0  E ii= 20 C 0 TJ 03 C "to  6  7  8  9  pH F i g . 18.  DNA-binding a c t i v i t y of P H I : 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 P H I containing about 25 units of UV-DNA-binding a c t i v i t y . After incubation for 10 min on i c e , each assay mixture was d i l u t e d with 1.7 ml of 100 mM NaCl and f i 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 s a l t 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 B u l l e t i n No. 106B).  Table V I I I .  E f f e c t of temperature on the DNA-binding a c t i v i t y of P H I .  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.  T a b l e IX.  Freeze-thaw  s t a b i l i t y of P H I .  DNA r e t a i n e d on f i l t e r , Experiment  I  II  Freeze-thaw treatment  UV-DNA  u-DNA  -  25.A  2.8  +  19.6  2.5  -  7.1  0.8  +  5.4  0.8  fmol  A l i q u o t s o f P H I were f r o z e n i n l i q u i d n i t r o g e n and thawed w i t h c o l d r u n n i n g water. The freeze-thaw procedure was r e p e a t e d f i v e times i n experiment I and t h r e e times i n experiment I I . The freeze-thaw t r e a t e d a l i q u o t s and u n t r e a t e d a l i q u o t s of P H I were then assayed f o r DNA-binding a c t i v i t y . The d a t a a r e the averages of d u p l i c a t e a s s a y s .  61  20 min Fig.  at  B O C  40  19. Heat s e n s i t i v i t y of P H I . A l i q u o t s o f P H I were heated a t 60 C f o r v a r i o u s p e r i o d s o f time and then assayed f o r DNA-binding a c t i v i t y w i t h UV-DNA ( • ) or u-DNA ( O ) a s t h e binding substrates. I n i t i a l l y , each a l i q u o t o f P H I c o n t a i n e d about 21 u n i 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 P H I to UV-DNA and u-DNA.  They were ATP, caffeine and sucrose.  In the presence of 10 mM ATP,  the binding of P H I to UV-DNA was  reduced by 40% and the binding of P H I to u-DNA was reduced to nearly zero CFig. 20).  In t h i s j e g a r d , P H I i s d i f f e r e n t from the uvrA  protein of Escherichia  coli  whose binding a c t i v i t y to UV-DNA i s  stimulated by the presence of ATP and GTP. Caffeine, which i s known to i n h i b i t DNA repair and bind to single-stranded regions of DNA (63, 64), i s an i n h i b i t o r of P H I binding (Fig. 21).  The i n h i b i t o r y e f f e c t of caffeine was greater with AAAF-DNA  and u-DNA than with UV-DNA.  3 mM caffeine inhibited the UV-DNA-  binding a c t i v i t y of P H I by about 35%, the AAAF-DNA-binding a c t i v i t y by nearly 70% and the u-DNA-binding a c t i v i t y by about 60%. experiment  In the  shown i n F i g . 21, the DNA was preincubated with caffeine  for 30 min before the addition of P H I .  Similar results were obtained  i n experiments where neither P H I nor DNA were preincubated with caffeine or where P H I rather than the DNA was preincubated with caffeine for 30 min.  I t i s l i k e l y that caffeine binds to DNA and thereby a l t e r s or  masks the DNA-binding s i t e s f o r P H I .  This i s suggested by the  observation of a d i f f e r e n t i a l e f f e c t of caffeine on the binding of P H I to AAAF-DNA and UV-DNA. The binding a c t i v i t y of P H I was also inhibited by sucrose. The presence of 5% sucrose i n the assay mixture reduced the UV-DNA-binding a c t i v i t y by h a l f and the u-DNA-binding a c t i v i t y to nearly zero.  On  the other hand, g l y c e r o l had l i t t l e effect on the DNA-binding a c t i v i t y of P H I (Table X).  Based on these observations, sedimentation  experiments  were performed with g l y c e r o l gradients rather than sucrose gradients.  Fig.  20.  E f f e c t o f ATP on t h e DNA-binding a c t i v i t y of P H I . DNA-binding a s s a y s were c a r r i e d out under t h e s t a n d a r d c o n d i t i o n s i n t h e p r e s e n c e of v a r i o u s c o n c e n t r a t i o n s of ATP. The b i n d i n g s u b s t r a t e s were UV-DNA (•) or u-DNA ( O ) . Each p o i n t i s t h e the average o f d u p l i c a t e a s s a y s .  25f  Caffeine, m M F i g . 21. E f f e c t of caffeine on the binding a c t i v i t y of P H I . Caffeine at the indicated concentrations was added to the standard assay mixture and incubated with UV-DNA ( • ) , AAAF-DNA (•) or u-DNA ( O ) f o r 30 min on i c e . Afterwards, P H I was added and the DNAbinding assays were carried out under the standard conditions.  Table X.  E f f e c t s 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 .  DNA retained on f i l t e r , fmol UV-DNA  u-DNA  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  % sucrose  The binding assays were carried out under the standard conditions except that the assay mixtures also contained various amounts of sucrose or glycerol as indicated.  The experiments described above were carried out at a saturating l e v e l of DNA damage.  At nonsaturating l e v e l of DNA damage, the  dependence of the DNA-binding a c t i v i t y of P H I on NaCl, MgCl , MnCl 2  2  or pH was s i m i l a r .  temperature,  However, the i n h i b i t o r y e f f e c t of  ATP on the binding a c t i v i t y of P H I to UV-DNA or u-DNA was more pronounced and am ounted to a 60% i n h i b i t i o n at 3 mM ATP when the DNA-binding assays were carried out with DNA UV-irradiated with 580 J/m and aliquots of P H I each containing 34 units of UV-DNA2  binding a c t i v i t y . 6. Glycerol gradient sedimentation analysis of P H I In order to get an estimate of the molecular weight of the DNA-binding protein, aliquots of P H I were analysed by sedimentation i n 10-30% g l y c e r o l gradients.  100 yg/ml of b a c i t r a c i n was included i n  the g l y c e r o l gradient solution to s t a b i l i z e the DNA-binding a c t i v i t y of P H I during the sedimentation. produced by Bacillus  lichenifovmis.  c l o s e l y related polypeptides.  B a c i t r a c i n i s an a n t i b i o t i c I t consists of a mixture of  The major component i s a c y c l i c  dodecapeptide c a l l e d b a c i t r a c i n A with a molecular weight of about 1,500  (65).  B a c i t r a c i n was chosen because of i t s low molecular weight.  A preliminary experiment Indicated that the presence of 100 yg/ml of b a c i t r a c i n i n P H I did not a f f e c t the DNA-binding a c t i v i t y of P H I . F i g . 22 shows that P H I sedimented  through a 10-30% glycerol  gradient containing 0.15 M NaCl with a sedimentation c o e f f i c i e n t of 2.0-2.5 S r e l a t i v e to the marker proteins.  A similar sedimentation  c o e f f i c i e n t was obtained when the centrifugation was carried out i n 0.5 M NaCl i n the gradient ( F i g . 23).  The r a t i o of the AAAF- or UV-DNA-  67  F i g . 22. Sedimentation v e l o c i t y analysis of P H I i n the presence of 0.15 M NaCl. A 200-yl aliquot of P H I i n 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 3.3% g l y c e r o l , 0.3 M NaCl and 100 yg/ml of 8-lactoglobulin was sedimented f o r 17 h through a 10-30% glycerol gradient containing 0.15 M NaCl as described i n Materials and Methods. 37 fractions were c o l l e c t e d . A 50-ul aliquot from each f r a c t i o n was assayed for binding a c t i v i t y towards UV-DNA ( • ) or u-DNA ( O ) . Marker proteins were: A, ovalbumin; B, cytochrome C. The sedimentation was from r i g h t to l e f t .  68  D  E  L P  c  0 TJ Q) C CD P CD  c_  < Z •  20  40  Fraction  F i g . 23. Sedimentation v e l o c i t y analysis of P H I i n the presence of 0.5 M NaCl. A 200-ul aliquot of P H I i n 10 mil Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 5% g l y c e r o l , 0.5 M NaCl and 100 ug/ml of B-lactoglobulin was sedimented for 27 h through a 10-30% l i n e a r glycerol gradient containing 0.5 M NaCl as described i n Materials and Methods. 35 f r a c t i o n s were collected. A 30-yl aliquot from each f r a c t i o n was assayed f o r binding a c t i v i t y 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 r i g h t to l e f t .  binding a c t i v i t y to the u-DNA binding a c t i v i t y of P H I increased from 10:1 i n the former experiment to 20:1 i n the l a t t e r experiment, indicating a further p u r i f i c a t i o n of the s p e c i f i c binding a c t i v i t y f o r AAAF-DNA and UV-DNA.  Assuming the protein i s spherical, the molecular  weight of P H I was estimated to be 20-25,000.  The recovery of the  DNA-binding a c t i v i t y from these g l y c e r o l gradients was greater than 90%. 7. Characterisation of the PIII-DNA complex Glass f i b r e f i l t e r s are commonly used to r e t a i n protein or DNA p r e c i p i t a t e s .  We therefore investigated the p o s s i b i l i t y of DNA  p r e c i p i t a t i o n i n our assays.  An aliquot of P H I containing 60 units  of UV-DNA-binding a c t i v i t y was incubated with UV-DNA under the standard conditions. min at- 10,000 g.  The assay mixture was then centrifuged for 10  I t was found that a l l the DNA remained i n  solution. PIII-DNA complex was also analysed by sedimentation through 10-30% g l y c e r o l gradients i n the presence of 0 mM NaCl, 50 mM NaCl and 150 mM NaCl.  Two major forms of PM2 DNA were resolved by v e l o c i t y  gradient sedimentation: the faster sedimenting covalently-closed c i r c u l a r supercoiled form and the nicked, relaxed c i r c u l a r form. Incubation with P H I did not induce any detectable changes i n the sedimentation p r o f i l e s of UV-DNA (Fig. 24-26).  P H I bound primarily  to the supercoiled form of UV-DNA but had much less binding a c t i v i t y towards the nicked form of PM2 DNA (Inserts, F i g . 24-26).  In the  absence of NaCl, there was very l i t t l e i f any d i s s o c i a t i o n of the bound PHI  from the supercoiled DNA (Insert, F i g . 24). In the presence of 50  mM NaCl, half of the bound P H I molecules dissociated from the UV-DNA during the 2 h of sedimentation (Insert, F i g . 25); and i n the presence  70  -DNA retained on f i l t e r % uv+ PHI • PHI  el  o  CN I  (14-17)  0.4 (14-17)  5.4 (20-22)  1.9 (20-22)  56.6  E a o  J 10  v  "\ 20  fr 30  Fraction  Fig.  24. Sedimentation of PIII-UV-DNA complex i n 10-30% glycerol and 0 mM NaCl. UV-DNA was incubated with an aliquot of P H I containing 80 units of UV-DNA-binding a c t i v i t y ( A ) or without P H I ( A ) under the standard conditions. 200 u l of the assay mixture was sedimented through a 10-30% glycerol gradient i n the absence of NaCl f o r 3 h as described i n Materials and Methods. The sedimentation was from right to l e f t . In the experiment where the UV-DNA was incubated with P H I , 35 fractions were c o l l e c t e d . In the experiment where the UV-DNA was incubated without P H I , 34 fractions were c o l l e c t e d . A 50-ul aliquot from each f r a c t i o n was was assayed for r a d i o a c t i v i t y . The background (25 cpm) was not subtracted. The remaining portions of the peak f r a c t i o n s 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 r e s u l t s were shown i n the i n s e r t . Numbers i n parentheses i n the i n s e r t indicated the f r a c t i o n numbers.  71  % uv--DNA retained on f i l t e r  + PHI  F i g . 25.  PHI  19.6 (20-23)  -0.2 (20-23)  3.8 (24-26)  0.5 (24-26)  Sedimentation of PIII-UV-DNA complex i n 10-30% g l y c e r o l and 50 mM NaCl. The experiment was performed as described i n the legend to F i g . 24 except that the g l y c e r o l gradient solution contained 50 mM NaCl i n addition to the other ingredients and the sedimentation was f o r 2 h. 34 fractions were collected f o r each sedimentation analysis.  72  % uv--DNA retained on f i l t e r  + PHI  A  Jl  8  PHI  2.5 (18-21)  -0.2 (17-20)  1.6 (23-25)  2.2 (21-23)  o  CN I  X  E a u  A  \A  10  20 Fraction  10  F i g . 26.  , + PI  20 Fraction  30  30  . — PI  Sedimentation of PIII-UV-DNA complex i n 10-30% g l y c e r o l and 150 mM NaCl. The experiment was performed as described i n the legend to F i g . 24 except that the g l y c e r o l gradient solution contained 150 mM NaCl i n addition to the other ingradients and the centrifugation was f o r 2 h. In the experiment where UV-DNA was incubated with P H I ( A ) , 36 f r a c t i o n s were c o l l e c t e d . In the experiment where the UV-DNA was incubated without P H I ( A ) , 34 fractions were c o l l e c t e d . To f a c i l i a t e comparison of the two sedimentation p r o f i l e s , the f r a c t i o n numbers of the l a t t e r experiment were plotted on a d i f f e r e n t scale.  of 150 mM N a C l , t h e d i s s o c i a t i o n was n e a r l y F i g . 26).  The e f f e c t o f s a l t  on t h e d i s s o c i a t i o n of P H I from  DNA was q u a l i t a t i v e l y s i m i l a r . the  f i n d i n g that  complete i n 2 h ( I n s e r t ,  These r e s u l t s do not c o n t r a d i c t  the s p e c i f i c binding  o f P H I t o UV-DNA was o p t i m a l  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 t h e f r e e m o l e c u l e s o f P H I from t h e DNA and t h e r e b y the  d i s s o c i a t i o n o f PIII-DNA complex.  Thus the b i n d i n g  UV-DNA was r e v e r s i b l e and d i d not i n v o l v e bonds between P H I and DNA. are  nicked  favored  of P H I to  the formation o f covalent  Covalent bonds between p r o t e i n s  formed by a d e n o v i r u s t e r m i n a l - p r o t e i n  topoisomerase I (66) and t h e DNA u n t w i s t i n g  (43,  and DNA  44), Escherichia  coli  enzyme o f r a t l i v e r  (67).  PIII-DNA complex was a l s o a n a l y s e d f o r undamaged DNA i n t h e absence o f N a C l .  Again, the binding  of P H I did not a l t e r the  s e d i m e n t a t i o n p r o f i l e o f t h e DNA ( F i g . 27) and t h e r e was a binding  preference for supercoiled  strong  DNA ( I n s e r t , F i g . 2 7 ) .  S e d i m e n t a t i o n a n a l y s e s f o r PIII-u-DNA complex i n t h e presence o f NaCl was  n o t performed. I n c u b a t i o n o f UV-DNA w i t h P H I f o r 45 min a t 37°C i n t h e presence  of 3 mM ATP, 7.5 mM M g C l  2  and 1 mM DTT a l s o d i d n o t induce any change  i n t h e s e d i m e n t a t i o n p r o f i l e of t h e DNA ( F i g . 28), the complex more s t a b l e The  (Insert, F i g . 28).  r e v e r s i b i l i t y of the binding  o f P H I t o UV-DNA was a l s o  from t h e c o m p e t i t i o n experiment shown i n F i g . 29. that P H I d i s s o c i a t e s The  nor d i d i t r e n d e r  from t h e UV-DNA v e r y s l o w l y  The r e s u l t s  evident  indicate  a f t e r i t i s bound.  amount o f t h e r a d i o a c t i v e PIII-UV-DNA complex was o n l y reduced by  % u-DNA retained on f i l t e r + PHI 24.2 (13-17)  1.0 (13-17)  4.4 (19-21)  2.9 (19-21)  20  10 Fra  -PHI  30  ction  F i g . 27. Sedimentation of PIII-u-DNA complex i n 10-30% g l y c e r o l and 0 mM NaCl. The experiment was performed as described In the legend to F i g . 24 with u-DNA incubated with (• ) or without P H I ( • ) . 34 fractions were collected f o r each sedimentation analysis.  75  % uv--DNA r e t a i n e d on f i l t e r  + A »  - PHI  PHI  18.3  (19-22)  0.1  (19-22)  4.2  (23-25)  2.5  (23-25)  1/ % #  XA  2  30  Fraction  F i g . 28.  Sedimentation o f PIII-UV-DNA complex formed i n t h e presence o f ATP and MgCl2« 139 fmol f o UV-DNA was i n c u b a t e d w i t h 80 u n i t s (UV-DNA b i n d i n g a c t i v i t y ) o f P H I ( A ) o r without P H I ( A ) i n 300-ul o f b u f f e r c o n t a i n i n g 10 mM T r i s - H C l (pH 7.5), 3 mM ATP, 7.5 mM M g C l and 1 mM DTT. A f t e r i n c u b a t i o n f o r 45 min a t 37°C, a 2 0 0 - y l a l i q o u t was s u b j e c t e d t o s e d i m e n t a t i o n a n a l y s i s as d e s c r i b e d 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 s e d i m e n t a t i o n analyses. 2  76  Fig.  29. R e v e r s i b i l i t y of the binding of P H I to UV-DNA. Aliquots of P H I 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/m ( • ) . Each point i s the average of duplicate assays. 2  40% when incubated with a 10-fold excess of unlabeled UV-DNA at 0 C for U  30 min.  Assuming f i r s t order k i n e t i c s , the d i s s o c i a t i o n can be  described by the equation: - In | = o where ^  k t  (2)  2  i s the rate constant for the d i s s o c i a t i o n of the PIII-UV-DNA  complex; t i s the time of incubation; B  q  i s the amount of PIII-DNA  complex at time 0; and B i s the amount of complex at time t. The -4 estimate L  f o r k^ i s about 3 x 10  -1 sec , which corresponds to a  h a l f - l i f e of the complex of about 40 min. 8. C a t a l y t i c a c t i v i t y No s i g n i f i c a n t amount of endonuclease or glycosylase a c t i v i t y was  detected i n aliquots of P H I under the various conditions  described i n Tables XI and XII. Incubation of UV- or u-DNA under the standard conditions with aliquots of P H I containing 80 units of UV-DNA-binding a c t i v i t y did not produce any material soluble i n 6% t r i c h l o r o a c e t i c a c i d .  UV-DNA,  u-DNA or l i n e a r PM2 DNA which had been incubated with P H I for 45 min at 37°C i n the presence of 10 mM T r i s - H C l , pH 7.5, 33 mM NaCl and 5 mM MgC^ also was not soluble i n 6% t r i c h l o r o a c e t i c acid.  Thus, under these two conditions, P H I does not exhibit any  exonuclease a c t i v i t y or behave l i k e c e r t a i n glycoproteins which s o l u b i l i z e DNA i n d i l u t e t r i c h l o r o a c e t i c acid without degrading the DNA (68, 69).  No ATPase a c t i v i t y was detected with aliquots of P H I  containing 50 u n i t s of UV-DNA-binding a c t i v i t y i n the presence of UV-DNA, u-DNA or single-stranded PM2 DNA.  Table XI.  Assay f o r DNA endonuclease a c t i v i t y .  % DNA nicked UV-DNA  u-DNA  +PIII  21.7  19.3  -PHI  19.7  18.8  Aliquots of 40 units (UV-DNA-binding a c t i v i t y ) of P H I were incubated f o r 30 min at 37 C with 139 fmol of UVDNA or u-DNA i n a 300 y l of reaction mixture containing 10 mM Tris-HCl, pH 7.5, 33 mM NaCl and 5 mM MgCl . Afterwards, assays f o r DNA endonuclease a c t i v i t y were performed as described i n Materials and Methods. The data are the averages of duplicate assays. 2  Table XII.  Assays for UV-DNA endonuclease and glycosylase a c t i v i t i e s under various conditions.  % UV-DNA nicked MgCl  2  DTT  ATP  endonuclease assay  mM  + PHI  PHI  glycosylase assay + 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 P H I each containing 40 units of UV-DNA-binding a c t i v i t y were incubated with 139 fmol of UV-DNA or u-DNA i n 300-ul reaction mixtures containing 10 mM Tris-HCl, pH 7.5, 33 mM NaCl and various concentrations of MgCl , DTT and ATP as indicated. Incubations were at 37 C f o r 30 min. Afterwards, 50-yl aliquots were assayed for DNA nicking or a l k a l i - l a b i l e s i t e s as described i n Materials and Methods. The amounts of DNA nicking and a l k a l i - l a b i l e s i t e s introduced by P H I 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. 2  It i s possible that P H I may unwind the DNA double h e l i x adjacent to the DNA binding s i t e .  We have therefore studied the e f f e c t of P H I  on the s u s c e p t i b i l i t y of UV-DNA and u-DNA to cleavage by the s i n g l e stranded  s p e c i f i c endonuclease from Neurospora  crassa.  This  endonuclease attacks supercoiled c i r c u l a r DNA, probably by recognizing some unpaired  regions i n the DNA (62, 70, 71).  It preferentially  nicked UV-irradiated DNA rather than unirradiated DNA (72). I t has been shown that the d e s t a b i l i z a t i o n of the DNA double h e l i x by the rep protein of Escherichia by the Neurospora  crassa  coli  f a c i l i a t e s cleavage of the DNA  endonuclease (73).  F i g . 30 shows that the  binding of P H I did not a l t e r the s u s c e p t i b i l i t y of UV- or u-DNA to cleavage by the Neurospora  crassa  endonuclease.  I t also appears that  the cleavage s i t e s for the endonuclease are not masked by P H I .  In our  experiment,, the rate of nicking of the UV-DNA by the endonuclease i s 3-4 f o l d higher than that of u-DNA. 9. DNA-binding proteins i n normal human and XP-fibroblasts DNA-binding proteins were also analysed  i n extracts of a normal  human f i b r o b l a s t and two XP-fibroblast c e l l l i n e s .  DEAE-fractions  were prepared from about 5-6 x 10^ c e l l s and contained  9-13 mg of  protein (Table X I I I ) . When the DEAE-fraction to phosphocellulose  of normal human f i b r o b l a s t s was subjected  chromatography, three major peaks of DNA-binding  a c t i v i t y were eluted from the column as with Hela c e l l s (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 e l u t i o n p r o f i l e was obtained with f i b r o b l a s t extract of the XP-group D c e l l l i n e , XP2NE.  F i b r o b l a s t s of the XP-group A c e l l l i n e ,  81  0.5  1  units/ml F i g . 30. E f f e c t of P H I on the s u s c e p t i b i l i t y of DNA to the single-stranded s p e c i f i c 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 a c t i v i t y ) of PHI ( A , A ) without P H I ( • , O ) under the standard conditions except that the assay mixtures also contained 100 ug/ml of acetylated BSA. The assay mixtures were then incubated f o r 30 min at 37 C with 5-yl aliquots of various concentrations of the single-stranded s p e c i f i c 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 f o r DNA nicking. Each point i s the average of duplicate assays. The Neurospora orassa endonuclease had an a c t i v i t y of 535 units/mg and was i n 3.2 M (NHi^SOit, pH 6.0. I t was diluted i n 10 mM Tris-HCl (pH 7.5) and 100 pg/ml of acetylated BSA p r i o r to experiment. o  r  82 Table 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 .  Volume of DEAE-fraction ml  Amount of protein i n DEAE-fraction mg  Cell line  C e l l number  207  4.8 x 10  7  7.1  8.5  2  5.1 x 10  7  7.1  9.2  3  6.0 x 10  8.5  12.9  8.0  9.4  1  XP5EG  XP2NE  XP5EG(2)'  t  5.0 x 10  7  A normal human c e l l l i n e . A XP-group A c e l l l i n e . A XP-group D c e l l l i n e . A repeated analysis with the c e l l l i n e XP5EG. F i g . 31 f o r d e t a i l s .  Read legend of  60 E  c o 0.A XI ca  C  3  5  4-1  c  01  A  0.2  u  c o u  <  / 1  Poo, 20  AO  4J iH CO  m  60  Fraction Fig.  31. Phosphocellulose chromatography of DNA-binding proteins from human f i b r o b l a s t extracts. Fibroblast extracts of the c e l l l i n e s (A) 207, (B) XP5EG and (C) XP2NE were subjected to analyses. The extracts were from c e l l s harvested at the 10th c e l l passage. (D) A repeated analysis f o r the c e l l l i n e XP5EG was performed with c e l l s harvested at the 13th passage. Each column f r a c t i o n was assayed with either UV-DNA (• ) or u-DNA ( o ) under the standard conditions. Phosphate concentration ( ••••).  o Salt  o concentration, M  XP5EG, seemed to be d e f i c i e n t 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 t h i s deficiency was reproducible.  The amounts of DNA-binding protein  (expressed as u n i t s of DNA-binding a c t i v i t y per mg of protein i n the DEAE-fraction)  which eluted at 375 mM potassium phosphate i n the normal  human f i b r o b l a s t s and XP f i b r o b l a s t s were similar to that of the phosphocellulose f r a c t i o n of P H I from Hela c e l l s (Table XTV). 10. Estimation of the equilibrium constant of the binding reaction and the concentration of P H I For the estimation of the equilibrium constant of the binding reaction and the concentration of P H I ,  we w i l l assume that one P H I  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 v a l i d i t y of these  two assumptions was already discussed i n an e a r l i e r section.  We w i l l  further assume that the binding reaction between P H I and the binding s i t e s on the UV-DNA are as follows: ± PS  P + The  d i s s o c i a t i o n e q u i l i b r i u m constant  calculated according  of t h e r e a c t i o n c a n be  t o the equation:  [P ] [ s ] f  f  (3) [PS] where P^ i s the concentration of the free P H I molecules,  S,. i s  the concentration of free binding s i t e s on UV-DNA, and PS i s the concentration of PIII-DNA complex. From equation  (3), i t can be shown that [S]  1 / 2  = K  d  + 1/2 [P]  (4)  T a b l e XIV.  Summary of the a n a l y s e s o f a UV-DNA-binding i n human f i b r o b l a s t e x t r a c t s .  U n i t s o f a c t i v i t y x 10  protein  per  mg o f p r o t e i n i n t h e D E A E - f r a c t i o n Fraction  Cell line  DEAE  Phospho-  UV-DNA  u-DNA  207  2.06  1.29  XP2NE  2.12  1.38  XP5EG  1.74  0.74  XP5EG(2)  1.90  0.70  207  0.96  0.21  XP2NE  0.82  0.10  XP5EG  0.88  0.10  XP5EG(2)  1.02  0.14  cellulose^  See t e x t and legend o f T a b l e X I I I f o r d e t a i l s cell  of the various  lines.  Peak f r a c t i o n s o f t h e DNA-binding a c t i v i t y which e l u t e d between 350-400 mM phosphate were pooled"and assayed i n d u p l i c a t e s f o r DNA-binding a c t i v i t y .  where [ S ] - ^  i s  t h e  concentration of binding s i t e s on UV-DNA that w i l l  saturate one-half of the P H I of P H I  molecules, and  i n the assay mixture (56).  [P] i s the concentration of  As shown i n F i g . 7,  [S]^^2  2 occurs at an UV-dose of about 250 J/m  .  From F i g . 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 s i t e for P H I .  Assuming a Poisson  d i s t r i b u t i o n of the binding s i t e s on the supercoiled DNA  and that the  average number of binding s i t e s per supercoiled DNA molecule increases 2 l i n e a r l y with an UV-dose up to 600 J/m  , we calculate that the average  number of binding s i t e s per molecule of supercoiled DNA  irradiated  with an UV-dose of 250 J/m  M of  2  i s 0.29.  With  3.7 x 1 0 ~  supercoiled DNA molecule i n the assay mixture, about 1.1 x 1 0 ~  10  10  [S]^^ i  s  therefore  M.  From the plateau of the dose response curve shown i n F i g . 7, one can estimate that the assay mixture contained about 23.5 of P H I  molecules.  [P] i s therefore 7.8 x 1 0  Using the values of t S ] ^ ^ equation  (A) to be about 7 x 10 ^  [F]»  - 1 1  fmol  M.  i s calculated from  M, which corresponds to a binding  energy of 13 kcal/mol.  Since the d i s s o c i a t i o n rate constant of the -A -1 binding reaction i s 2.9 x 10 sec as has been estimated from F i g . 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 P H I  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).  I t i s much lower than  the Kj values f o r the binding of lac repressor to nonoperator which i s i n the order of 10 ^ M (7A), the noncooperative  DNA  binding of  gene 32 protein of phage T4 to duplex DNA which i s i n 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 i n the order of  10~  10  M (75) and the cooperative binding of the gene D5 protein to duplex DNA which i s '6.27 x 1 0 ° M _ 1  (76)  We had used 10 y l of P H I  i n the experiment depicted i n F i g . 7.  The results of the experiment suggests that there were 23.5 PHI  molecules per 10 y l of P H I .  The concentration of P H I  fmol of was  _9 therefore 2.35 for P H I ,  x 10  M.  and i f a l l P H I  the amount of P H I  I f our p u r i f i c a t i o n scheme had a 30%  recovery  molecules were extracted from the Hela c e l l s ,  per Hela c e l l was  i n the order of 10^  molecules.  Discussion i . Advantages of using glass f i b r e f i l t e r s i n the f i l t e r - b i n d i n g assay We have used GF/C f i l t e r s i n the f i l t e r - b i n d i n g assay of  PHI.  Conventionally, n i t r o c e l l u l o s e membrane f i l t e r s are used f o r 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 n i t r o c e l l u l o s e f i l t e r s have the disadvantage  that they also bind single-stranded DNA  and to a c e r t a i n extent DNA  with h e l i c a l d i s t o r t i o n s (49, 82).  To reduce a high background due to  the retention of these forms of DNA  i n the f i l t e r - b i n d i n g assays,  i t i s necessary to treat the n i t r o c e l l u l o s e f i l t e r s i n an a l k a l i solution followed by extensive washing and n e u t r a l i z a t i o n of the f i l t e r s (42, 77, 80, 81).  Glass f i b r e 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 n i t r o c e l l u l o s e f i l t e r s , glass f i b r e f i l t e r s are  more convenient to use.  The glass f i b r e f i l t e r s can be used d i r e c t l y  by wetting them b r i e f l y with the f i l t r a t i o n buffer, whereas the n i t r o c e l l u l o s e 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  i n the f i l t e r binding assays with glass f i b r e f i l t e r s .  Generally,  f i l t e r - b i n d i n g assays were performed with f i l t e r speeds of less than 5 ml/min with the n i t r o c e l l u l o s e f i l t e r s (36, 57, 77).  We have  demonstrated that i n our f i l t e r - b i n d i n g assay, the retention of PIIIDNA  complex i s 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 f i b r e f i l t e r s for assaying the DNA-binding a c t i v i t y 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 f i b r e f i l t e r s i n the f i l t e r - b i n d i n g assays.  They  reported that the e l u t i o n of the adenovirus DNA-terminal protein complex with sodium dodecyl sulphate from the glass f i b r e was  filters  50-fold more e f f i c i e n t compared with the n i t r o c e l l u l o s e f i l t e r s . In conclusion, we think the f i l t e r - b i n d i n g assay using  f i l t e r s i s a simple, s e n s i t i v e and reproducible assay for  GF/C  DNA  binding proteins. 2. Mechanism of retention of PIII-DNA complex by the GF/C  filters  We do not understand the mechanism by which the glass f i b r e f i l t e r s r e t a i n the PIII-DNA complex. We are aware that the glass f i b r e f i l t e r s are commonly used to r e t a i n protein or DNA denaturants. not due to DNA was  precipitated by t r i c h l o r o a c e t i c acid or other  However, the retention of the PIII-DNA complex was p r e c i p i t a t i o n or intermolecular DNA  concluded from the sedimentation  This  analysis of the PIII-DNA complex  through the g l y c e r o l gradients, since DNA a s i m i l a r sedimentation  aggregration.  clearly  complexed with P H I  c o e f f i c i e n t as free  had  DNA.  We .think that the i n t e r a c t i o n 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 f i b r e s (Glass microfibre f i l t e r s , Whatman Publication No.  824).  In f a c t , GF/C  antigen-antibody  f i l t e r s have been used to r e t a i n soluble  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 P H I 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 f i b r e f i l t e r s with pore sizes greater than 1.5 ym are l e s s e f f i c i e n t i n r e t a i n i n g the PIII-DNA complex.  This r e s u l t may indicate that the hydrodynamic diameter  of the PIII-DNA complex i s l e s s than 1.5 ym. I t i s however d i f f i c u l t to envisage that the binding of one or a few molecules of P H I can bring about a d r a s t i c change i n the hydrodynamic diameter of a PM2 DNA molecule without a s h i f t i n the sedimentation c o e f f i c i e n t of the DNA. A l t e r n a t i v e l y , GF/C f i l t e r s with smaller pore sizes might be more e f f i c i e n t because of a higher content of surface materials which interact with the P H I molecules. 3. Comparison of P H I with other UV- or AAAF-DNA-bind ing proteins from human c e l l s P H I i s l i k e l y to be d i f f e r e n t from an AAAF-DNA-binding protein p u r i f i e d from human placenta by Moranelli and Lieberman (42). The l a t t e r protein binds e f f i c i e n t l y to l i n e a r duplex T7 DNA treated with AAAF, MMS and MNUA but does not recognise UV-irradiated DNA. P H I , on the other hand, binds e f f i c i e n t l y to supercoiled PM2 DNA treated with UV, AAAF, MNNG but not MMS. P H I i s also d i f f e r e n t from a UV-DNA-binding protein that has been p u r i f i e d from human placenta by Feldberg and Grossman (40, 41). The l a t t e r UV-DNA binding protein binds e f f i c i e n t l y to linear DNA treated with nitrous acid and sodium b i s u l p h i t e . Whether i t binds to DNA treated with AAAF or MNNG has not been reported. binding protein elutes from the phosphocellulose  The UV-DNA  column at around  0.175  M potassium phosphate, and has a molecular weight greater than  100,000.  P H I , 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 s i m i l a r i t i e s between PIII and the UV-DNA unwinding protein from CLL lymphocyte extracts (28).  The molecular weight of  the unwinding protein i s 24,000, which i s about the same as PIII. Both proteins are eluted from an UV-irradiated DNA-cellulose 1 M NaCl.  column at  The binding substrate s p e c i f i c i t y of the unwinding protein  has not been reported.  However, the two proteins may d i f f e r i n  t h e i r binding a f f i n i t i e s to single-stranded DNA. binds t i g h t l y to single-stranded DNA-cellulose NaCl for e l u t i o n .  The unwinding protein  column and required 2 M  On the other hand, PIII appears to have a weaker  a f f i n i t y for single-stranded DNA than UV-DNA. In f a c t , to our knowledge, the only other n a t u r a l l y occuring protein which binds to DNA treated with UV and AAAF i s the gene 32 protein of phage T4 (cited i n reference 84). 4. B i o l o g i c a l s i g n i f i c a n c e of PIII We i n f e r from the abundance of PIII, which i s probably 10"* molecules per Hela c e l l , that i t must be important Two  i n DNA metabolism.  observations suggest a possible r o l e of PIII i n DNA r e p a i r .  F i r s t , the binding a c t i v i t y of PIII i s damage-dependent.  Second,  the binding of PIII to UV- or AAAF-DNA has a small d i s s o c i a t i o n equilibrium constant of 7 x 10 *" M which indicates a strong a f f i n i t y -  1  of PIII to UV- or AAAF-DNA damage.  However, d i r e c t  for a repair function of PIII i s s t i l l lacking.  evidence  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 o f t h e r e 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, a t s i m i l a r l e v e l s as t h a t i n normal human f i b r o b l a s t s .  Further, P H I  does n o t p o s s e s s any s i g n i f i c a n t DNA endonuclease, ^ g l y c o s y l a s e , e x o n u c l e a s e or ATPase a c t i v i t y .  An argument a g a i n s t a r o l e of P H I  i n the e x c i s i o n r e p a i r of pyrimidine  dimers i s t h e requirement o f  h i g h UV-dose f o r the c r e a t i o n o f o n l y a l i m i t e d number o f b i n d i n g s i t e s f o r P H I on t h e DNA.  With a UV-dose o f 600 J/m , about 45% 2  of t h e DNA m o l e c u l e s have a b i n d i n g 60  thymidine dimers a r e i n t r o d u c e d  s i t e f o r P H I w h i l e more than p e r DNA m o l e c u l e s (85).  There i s  a l s o t h e p o s s i b i l i t y t h a t P H I might a c t u a l l y be a p r o t e i n i n DNA r e p l i c a t i o n o r t r a n s c r i p t i o n . creat unnatural  PHI  treatments  could  DNA-binding s i t e s f o r P H I .  Nevertheless, each other  UV o r other  involved  recalling  t h a t t h e uvrA, B and C p r o t e i n s  complement  t o form a UV-endonuclease a c t i v i t y , i t i s p o s s i b l e  that  may o n l y r e v e a l i t s r e p a i r f u n c t i o n i n the presence o f other  proteins. I t i s a l s o i n t e r e s t i n g t h a t P H I b i n d s e f f i c i e n t l y t o MNNG-DNA but n o t MMS-DNA . 1  MNNG a l k y l a t e s t h e DNA v i a a. S. 1 r e a c t i o n w h i l e N T  MMS a l k y l a t e s t h e DNA p r i m a r i l y v i a a S 2 r e a c t i o n (86, 87).  MNNG,  N  a N-nitroso  a l k y l a t i n g agent, has a h i g h e r  n u c l e i c a c i d s than MMS (87).  a f f i n i t y f o r oxygen i n  One o f t h e a l k y l a t e d s i t e s on t h e DNA  t r e a t e d w i t h MNNG i s t h e O ^ - p o s i t i o n  o f the guanine r e s i d u e  (86).  The  6 0 -alkylguanine  residue  can b a s e - p a i r  w i t h a thymine r e s i d u e  DNA, and r e s u l t s i n a GC t o AT t r a n s i t i o n m u t a t i o n (88).  1  i n the  There i s  I r i t h i s d i s c u s s i o n , we assume t h a t the p r o t e i n which b i n d s t o  AAAF-DNA and UV-DNA a l s o b i n d s t o MNNG-DNA and u-DNA.  a good c o r r e l a t i o n between the persistence of 0°-alkylguanine i n brain tissue and the frequency of occurence of brain tumors i n 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 d e f i c i e n t (6, 91).  Thus, MNNG may be  similar to UV and AAAF i n 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 c e l l s .  In other words, the repair  process of c e r t a i n 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 c e l l s (92).  of the rat l i v e r  The l a t t e r result suggests that the pretreatment  may have  induced or activated a process or a s p e c i f i c enzyme which i s involved 6 i n the repair of 0 -alkylguanine and DNA damage induced by acetylaminofluorene. The dependence of the binding a c t i v i t y of PIII on DNA supercoiling suggests that PIII might unwind the DNA h e l i x .  The higher free energy  of a supercoiled DNA than an nonsupercoiled DNA favors the binding of proteins which can unwind the DNA double h e l i x and thereby reduce the number of superhelical turns (61).  The difference i n the equilibrium  constants for the binding of an unwinding protein to superhelical and nonsuperhelical DNA can be very large (93).  However, we have  f a i l e d to detect an unwinding effect of PIII with the single-stranded s p e c i f i c endonuclease from Neurospora  crassa.  I t remains to be  tested i f PIII w i l l enhance the UV-endonuclease a c t i v i t y of luteus  Micrococcus  i n a s i m i l a r way as the UV-DNA-unwinding protein of the CLL  lymphocytes. 5. Nature of the binding s i t e for PIII A l i k e l y feature of the DNA binding s i t e for PIII i s s i n g l e -  strandness which i s suggested  from the a f f i n i t y of P H I to s i n g l e -  stranded DNA and the i n h i b i t i o n of P H I by caffeine.  Local denatured  regions are known to be present i n DNA treated with UV- or AAAF (94-96).  Native supercoiled PM2 DNA also contains regions with  unpaired bases (at least t r a n s i e n t l y ) , p a r t i c u l a r l y 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 P H I .  Instead of  A+T-rich regions, hairpin or cruciform structures (70, 98, 99) may be important  f o r the formation of binding s i t e s for P H I .  suggested  that the base-pairing i n a hairpin structure i s p r e f e r e n t i a l l y  disrupted by DNA damage (70).  I t has been  But single-strandness alone apparently  i s not enough f o r an e f f i c i e n t binding by P H I , since single-stranded DNA i s not bound e f f i c i e n t l y by P H I as compared with the supercoiled DNA damaged with UV, AAAF or MNNG.  Thus, DNA supercoiling i s required  for an e f f i c i e n t binding of P H I to DNA damage. The PM2 DNA isolated from natural sources i s composed of a Boltzmann d i s t r i b u t i o n of geometrical isomers which d i f f e r from each other by an i n t e g r a l number of superhelical turns (100, 101). There are on the average about 90 superhelical turns i n 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 p a r t i c l e (103, 104). The superhelical density may influence the i n t e r a c t i o n 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. A l t e r n a t i v e l y , DNA supercoiling might be necessary for the formation or s t a b i l i z a t i o n of the DNA-recognition  s i t e f o r a protein. The  96 s u s c e p t i b i l i t y of some supercoiled DNA to c e r t a i n single-stranded s p e c i f i c nucleases i s known to be dependent on the degree of DNA supercoiling (61, 70, 98, 99). For example, PM2 DNA molecules are not s e n s i t i v e to the Neurospora crassa  endonuclease unless they have  superhelical densities higher than -0.029 (70).  The nuclease-sensitive  s i t e s on the supercoiled DNA may be s p e c i f i c and seem to l i e i n the loops of p o t e n t i a l cruciform structures (99).  The cruciform structures  may be more stable i n DNA with high supercoiled densities and i n A+Tr i c h regions (98, 99). I t i s thus possible that the damage-induced binding 6 i t e s of PIII might only be formed on those PM2 DNA molecules with superhelical densities higher than a c e r t a i n value.  Studies of  the DNA-binding a c t i v i t y of PIII with DNA of d i f f e r e n t defined superhelical densities should allow us to determine whether there exists a minimal superhelical density above which the binding s i t e s f o r PIII are r e a d i l y induced by DNA damage. There i s another interesting p o s s i b i l i t y f o r the nature of the binding s i t e s 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 s u b s t i t u t i o n at the 8-position of the guanine residue (95).  UV can also introduce  adduct to the 8-position of the purine residues i n the DNA.  A specific  endonuclease a c t i v i t y directed towards the UV-induced 8-alkylated purines i n the PM2 DNA was i d e n t i f i e d i n extracts of luteus,  which probably recognised the syn conformation  residues (105).  Micrococcus of the purine  We plan to determine i f PIII w i l l recognise  97 the UV-induced 8-alkylated purine residues. conformation  Interestingly, the syn  of the guanine residues i s preferred i n the left-handed  Z-DNA structure (106).  A DNA structure which i s probably assumed by  poly(dG-dC)'poly(dG-dC) at s a l t concentration higher than 2.5 M NaCl (107).  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