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Partial purification and characterisation of apurinic endonuclease activity from Hela cells Tsang, Siu Sing 1978

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PARTIAL PURIFICATION AND CHARACTERISATION OF APURINIC ENDONUCLEASE ACTIVITY FROM HELA CELLS, by SlU SINGfSANG B.So, M c G i l l U n i v e r s i t y , Montreal, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Genetics Program) We accept t h i s t h e s i s as conforming t o the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1978 (c) Siu Sing /Tsang, 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of GENET I CS  The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date 4 ' Oct., 1978. ABSTRACT A p u r i n i c endonuclease a c t i v i t y in human f i b r o b l a s t s had been p r e v i o u s l y r e s o l v e d i n t o af low-through and a h i g h - s a l t e l u a t e spe-c i e s by phosphocellulose chromatography (Kuhnlein, U. e t a l . , Nucl. A c i d . Res. 5: 951-960, 1978). Enzyme a c t i v i t y in the flow-through s p e c i e s amounted t o 20-30% t h a t of the h i g h - s a l t e l u a t e s p e c i e s . The flow—through enzyme species was not found i n - e e l I l i n e s of xeroderma pigmentosum complementation group D. In t h i s t h e s i s , a p u r i n i c endonuclease a c t i v i t y was analysed in Hela c e f l l s . S p e c i f i c enzyme a c t i v i t y in crude e x t r a c t s "of Hela c e l l s was i n the range of 400-800 units/mg p r o t e i n , s i m i l a r t o t h a t of , human f i b r o b l a s t s which was between 380-680 units/mg p r o t e i n . Three sp e c i e s of endonuclease a c t i v i t y f o r a p u r i n i c DNA were resolve d by phosphocellulose chromatography. They were designated as Peak I, Peak I f , and Peak I I I . Peak I d i d not adsorb t o the phosphoceIIuIose column at' 10 mM KP0 4 (pH 7.4) (flow-through a c t i v i t y ) , Peak II e l u t e d from t h e column a t about 210 mM KP0 4 (pH 7.4) and Peak I I I a t 260 mM KPO^ (pH 7.4). Based on t h e i r a f f i n i t y t o phosphoceI IuIose, we pre-sumed Peak I and Peak III corresponded t o the flow-through and high-s a l t e l u a t e s p e c i e s in human f i b r o b l a s t s r e s p e c t i v e l y . Under our ex-perimental c o n d i t i o n s , the flow-through enzyme a c t i v i t y in both Hela c e l l s and normaS human f i b r o b l a s t s was only 2-4% of the a c t i v i t y of h i g h - s a l t e l u a t e s p e c i e s . We suspect t h a t t i s s u e c u l t u r e c o n d i t i o n s may a f f e c t the c e l l u l a r l e v e l of the flow-through species of a p u r i n i c endonuclease. Peaks l - l I f were o p t i m a l l y a c t i v e a t pH 7.5-8.0 and 5-10 mM MgCI, I I I . They were 'inhibited by i n c r e a s i n g concentrations of KCI and NaCI except Peak III which was s l i g h t l y s t i m u l a t e d by 20-40 mM KCI. The three species were d i s t i n g u i s h e d by t h e i r t h e r m o s e n s i t i v i t i e s in a 50 mM KPO. b u f f e r . Peak I was s t a b l e at 45°C. Peak III was h e a t - l a b i l e , having a h a l f - l i f e of 2-3 min at 45°C. Peak II seemed to contain two components, one with a ha I f - l i f e of 2-3 min at 45°C, and the other with a h a l f - l i f e of 25 min. In human f i b r o -b l a s t s , both the flow-through and h i g h - s a l t e l u a t e species of apu-r i n i c endonuclease were reported t o be s t i m u l a t e d t o 2.5-fold by 10 mM KCI. They had a ha I f - l i f e of 6 min at 45°C in a 230 mM KP0 4 (pH 7.4) b u f f e r . Thus, Peaks I - l I I and enzyme species from human 2+ f i b r o b l a s t s had a s i m i l a r pH optimum, and Mg requirement, but they d i f f e r e d in t h e i r t h e r m o s e n s i t i v i t i e s and i n h i b i t i o n by higher s a l t c o n c e n t r a t i o n . We do not know as yet whether these d i f f e r e n c e s r e f l e c t the n e o p l a s t i c nature of Hela c e l l s o r the d i f f e r e n t t i s s u e o r i g i n s of Hela c e l l s and human f i b r o b l a s t s . When e i t h e r Peak I o r Peak I I I was rechromatographed on the phosphoceI Iulose column, a c t i v i t y was recovered in both the flow-through and h i g h - s a l t e l u a t e f r a c t i o n s . The r e s u l t suggested an i n t e r c o n v e r s i o n phenomenon between the flow-through and h i g h - s a l t e l u a t e species of a p u r i n i c endonuclease, This was f u r t h e r supported by molecular weight determinations of the a p u r i n i c endonucI eases In Peaks l - l I I. A p u r i n i c endonuclease a c t i v i t y in Peak III and Peak II had a molecular weight of 35,000-40,000 and 22,000-25,000 respective-l y . Peak I had two components with molecular weights s i m i l a r t o those of Peak II and Peak I I I . An understanding of the conversion i v. between the d i f f e r e n t a p u r i n i c endonuclease species may help in e l u c i d a t i n g the molecular defects of xeroderma pigmentosum comple-mentation group D. A p u r i n i c endonuclease a c t i v i t y in Peaks l - l I I was found to be a s s o c i a t e d with a high molecular weight complex. The complex could be d i s s o c i a t e d by high s a l t treatment. The p o s s i b l e b i o l o g i c a l s i g n i f i c a n c e of the high mo I ecu Iar weight complex Is discussed. We a l s o found t h a t a p u r i n i c endonuclease could adsorb to the Sephadex g e l . The adsorption would lead t o an aberrant e s t i m a t i o n of molecular weight of the p r o t e i n . The problem was solved with an e l u t i o n b u f f e r of high i o n i c s t r e n g t h . V . TABLE OF CONTENTS Page ABSTRACT 11 TABLE OF CONTENTS " v LIST OF TABLES ' v! TI LIST OF FIGURES ix ACKNOWLEDGEMENT x i i ABBREVIATIONS x i I I INTRODUCTION 1 1. A p u r i n i c / a p y r i m i d i n i c s i t e s as a common DNA l e s i on 1 2. P o s s i b l e c e l l u l a r e f f e c t s of a p u r i n i c / a p y r i m i d i n i c s i t e s 2 3. DNA r e p a i r mechanisms 3 4. A p u r i n i c endonuclease in human c e l l l i n e s 8 5. I nhe r i ted DNA r e p a i r de fec t s i n xeroderma pigment-osum 8 6. Ob jec t i v e s 11 MATERIALS AND METHODS 1. T i s sue cu I t u r e 14 (a) Cel I I ines 14 (b) C u l t u r e media . 14 (c) Cel I growth 14 (d) Cel I ha rves t i ng 14 2. P repa ra t i on of PM2 phage DNA 15 (a) B a l - b r o t h 15 (b) B a l - t op agar ' 15 v i . Page (c ) Ba I - p l a t e s ; 15 (d) P l a q u e a s s a y f o r phage t i t e r • 15 ( e ) P r e p a r a t i o n o f phage s t o c k 16 ( f ) P r e p a r a t i o n o f 3 H - l a b e l l e d PM2 DNA 16 3. Enzyme p u r i f i c a t i o n 17 (a ) P r e p a r a t i o n o f c e l l e x t r a c t 17 (b) D E A E - c e l I u l o s e ch r omatog r aphy 20 Cc) Phosphoce I I u I o se ch romatog raphy 20 4. PM2 DNA d e p u r i n a t i o n 21 5. F i l t e r b i n d i n g a s s a y 21 6. A p u r i n i c e n d o n u c l e a s e a s s ay 23 7. Sephadex G-100 co lumn ch romatog raphy 24 8. S u c r o s e g r a d i e n t c e n t r i f u g a t i o n 25 9. S a l t - t r e a t m e n t o f enzyme a l i q u o t s 25 10. P r e p a r a t i o n o f a c e t y l a t e d BSA 26 11. P r o t e i n d e t e r m i n a t i o n s 26 R e s u l t s 27 1. Q u a n t i f i c a t i o n o f number o f a p u r i n i c / a p y r i m i d i n i c s i t e s i n d e p u r i n a t e d DNA 27 2. P u r i f i c a t i o n o f a p u r i n i c e n d o n u c l e a s e a c t i v i t y f r o m H e l a e e l Is 27 ( a ) H i g h - s p e e d c e n t r i f u g a t i o n 27 (b ) D E A E - c e l I u l o s e ch r omatog r aphy 31 ( c ) P h o s p h o c e l I u l o s e ch r omatog r aphy 31 3 . P h o s p h o c e l I u l o s e r e c h r o m a t o g r a p h y o f a p u r i n i c e n d o -n u c l e a s e a c t i v i t y i n H e l a c e l l s 33 V Page 4. G e n e r a l p r o p e r t i e s of a p u r i n i c e n d o n u c l e a s e a c t i v i t y i n H e l a e e l Is , 34 (a) Requirement o f magnesium i o n s 34 (b) pH optimum 34 (c) E f f e c t s o f NaCl and KCI c o n c e n t r a t i o n 38 (d) Heat i n a c t i v a t i o n 38 5. M o l e c u l a r w e i g h t d e t e r m i n a t i o n s o f P e a k s ' I - M l ' .... 38 D i s c u s s i o n 59 1. Comparison o f a p u r i n i c e n d o n u c l e a s e a c t i v i t y i n H e l a c e l l s and human f i b r o b l a s t s , 59 (a) G e n e r a l p r o p e r t i e s " 59 (b) R e l a t i v e p r o p o r t i o n o f f l o w - t h r o u g h and h i g h -saM^ e l u a t e s p e c i e s o f a p u r i n i c e n d o t i u c l e a s e a c t i v i t y 60 2. I n t e r c o n v e r s i o n o f f l o w - t h r o u g h and h i g h - s a l t e l u a t e s p e c i e s o f a p u r i n i c e n d o n u c l e a s e from H e l a c e II s 61 3. Mo I ecu I a r w e i g h t d e t e r m i n a t i o n s o f Peaks 1 — I I I ... 62 LIST OF TABLES Page Table 1. P u r i f i c a t i o n of a p u r i n i c endonuclease a c t i v i t y f nom He I a ce I I s 30 i x . LIST OF FIGURES Fiqure Page 1 - Schematic r e p r e s e n t a t i o n of the p r i n c i p l e mech-anisms f o r the r e p a i r of i n t r a s t r a n d p y r i m i d i n e dimers i n DNA 4 2 Base e x c i s i o n DNA r e p a i r mechanism f o r the r e p a i r of p a r t l y deaminated DNA 5 3. I s o l a t i o n of PM2 phage p a r t i c l e s by cesium c h l o -r i d e - d e n s i t y - g r a d i e n t e q u i l i b r i u m c e n t r i f u g a t i o n 18 4 U l t r a v i o l e t absorption spectrum of p u r i f i e d PM2 phage DNA in 10 mM Tr i s - H C l (pH 7.5) 19 5 S t a n d a r d i z a t i o n of f i l t e r - b i n d i n g assay 22 6 Time course of a l k a l i h y d r o l y s i s of a p u r i n i c PM2 DNA 28 7 Time course of depurination of PM2 DNA at 70°C 29 8 PhosphoceI IuIose chromatography of a p u r i n i c endo-nuclease a c t i v i t y from a Hela c e l l s e x t r a c t 32 9 - Phosphocellulose rechromatography of Peak I .... 35 1 0 PhosphoceI Iulose rechromatography of Peak III 35 11 E f f e c t of MgC^ on a p u r i n i c endonuclease a c t i v i t y of Hela c e l Is 36 12 E f f e c t of pH on a p u r i n i c endonuclease a c t i v i t y of Hela c e l Is 37 ; 13: ... R e l a t i v e a c t i v i t y of (a) Peak I, (b) Peak II and (c) Peak I I I a t d i f f e r e n t NaCI concentrations . 39 Figure . Page 14 R e l a t i v e a c t i v i t y of (a) Peak I,; Ob) Peak I I and (c) Peak II I at d i f f e r e n t KCI concentrations ... 40 15 Heat i n a c t i v a t i o n curve of a p u r i n i c endonuclease a c t i v i t y in Peaks l - l I I ,. 41 16 Sephadex G-100 chromatography of Peak I c 43 17- ." Sephadex G-100 chromatography of Peak III 44 18- Sephadex G-100 chromatography of a Hela DEAE poo I 46 19 Sephadex G<-100 chromatography of flow-through, a p u r i n i c endonuclease a c t i v i t y p u r i f i e d from Peggy ce I I s 47 20- . Sephadex G-100 chromatography of "low molecular weight "form of Hela a p u r i n i c endonuclease. 48 21 Sucrose g r a d i e n t c e n t r i f u g a t i o n of a Hela DEAE pool 49 22 Sucrose g r a d i e n t c e n t r i f l i g a t i o n of a Hela DEAE pool which had been s a l t - t r e a t e d with 2M KCI before c e n t r i f u g a t i o n 51 23 Sephadex G-100 chromatography of the peak f r a c t i o n s of a p u r i n i c endonuclease a c t i v i t y obtained, in the experiment-described. in Figure 22. •. 52 . 24 C a l i b r a t i o n of Sephadex G-100 column 53 - 25 Sephadex G-100 chromatography of s a l t - t r e a t e d Peak HI w i t h e l u t i o n b u f f e r Y 54 26 Sephadex G-100 chromatography of s a l t - t r e a t e d Peak II w i t h e l u t i o n b u f f e r Y 56 Fiqure F'age 27 SepHadex G-100 chromatography of : s a I t - t r e a t e d Peak I with e l u t i o n b u f f e r Y ... 57 ACKNOWLEDGEMENT I am g r a t e f u l to both Dr. U. Kuhnlein and Dr. H.F. S t i c h f o r t h e i r s u p e r v i s i o n and int r o d u c i n g me t o the f a s c i n a t i n g f i e l d of DNA r e p a i r and environmental c a r c i n o g e n e s i s . Dr. L.D. Skarsgard k i n d l y provided laboratory space and various f a c i l i t i e s . I thank my colleagues f o r t h e i r s t i m u l a t i n g d i s c u s s i o n and k i n d l y a s s i s t a n c e , e s p e c i a l l y Miss J . Edwards, Miss 0. Yu and Mrs. W. S t i c h . Studentship awards from the Medical Research Council (Aug., '1976 - March, 1978) and the National Cancer I n s t i t u t e of Canada ( A p r i l , 1978 - ) are acknowledged. F i n a l l y , I am indebted to my w i f e , Francoise, whose thought-f u l n e s s , patience, encouragement and help have made t h i s t h e s i s a r e a I i t y . x i i i . ABBREVIATIONS AMV avian m y e l o b l a s t o s i s v i r u s ? BSA bovine serum albumin Ci C u r i e , one Cur i e = the q u a n t i t y of a r a d i o a c t i v e '• isotope undergoing 3.7x10^ d i s i n t e g r a t i o n s per sec. CsCI cesium c h l o r i d e DEAE-cellulose 0-(diethyI aminoethyI) c e l l u l o s e DTT d i t h i o t h r e i t o l DNA d e o x y r i b o n u c l e i c a c i d EDTA ethyIenediaminetetracetate KP0 4 potassium phosphate b u f f e r , made up of d i b a s i c potassium phosphate(t^HPO^) and monobasic potassium phosphate (KH2P04.) MOI m u l t i p l i c i t y of i n f e c t i o n O.D. o p t i c a l d e n s i t y o r absorbance P0P0P 1,4-bis (2-(5-phenyloxyazolyI))-benzene POP 2,5, diphenyloxazole SDS sodium dodecyIsuIphate T r i s tris(hydroxymethyI) amincmethane UV I i g h t u l t r a v i o l e t I i g h t 1. INTRODUCTION T. A p u r i n i c / a p y r i m i d i n i c sites.-as common DNA le s i o n s : Perhaps one of the most common forms of DNA'damage i s the loss o f pu r i n e and pyrimidine bases from the DNA. These processes, d e p u r l n a t i o n and de p y r i m i d i n a t i o n , involve breakage of the g l y c o -s f d i c band between the purine o r pyrimidine bases and the deoxy-r i b o s e m o i e t i e s of the DNA. Purine and py r i m i d i n e bases have been demonstrated t o be re-leased Err d e t e c t a b l e q u a n t i t i e s from DNA at neutral pH and 70°C (1) . The i n i t i a l rate constant of depurination i s 2.4 x 10 ^sec \ Depyrfmidination i s about 10-20 times slower than depurination (2) . At p h y s i o l o g i c a l c o n d i t i o n s of 37°C and pH 7.4, the in v i v o rate constant of depurination has been estimated t o be in the o r -- 9 - 1 der o f 2 x 10 min DMA modifi e d by some chemical or ph y s i c a l agents has a much higher dlepurination/depyrimidination rate ( 3 ) . An example i s a l -k y l a t e d DNA. A l k y l a t i o n of DNA r e s u l t s in the formation of purine and pyrrtttidine d e r i v a t i v e s with l a b i l e g l y c o s i d i c bonds (4,5). Some mod i f i e d bases, such as 3-methyIadenine and 0^-methyIguanine can a l s o be removed by s p e c i f i c DNA-gIycosidases (6,7). The in v i v o d e p u r i n a t i o n r a t e constants of 7-methyIguanine and 3-methyIadenine -4 -1 -3 -1 are o f the o r d e r of 1 x 10 min and 4 x 10 min r e s p e c t i v e l y (8). F i n a l l y , a p y r i m i d i n i c s i t e s are formed during the process of removal of u r a c i l residues from the DNA. U r a c i l residues are 2. introduced i n t o the DNA as a r e s u l t of deamination of c y t o s i n e residues (9,10). U r a c i l can a l s o be incorporated i n t o DNA in ptaee of thymine during rep Ii c a t i o n • ' (11,12). The enzyme t h a t i s thought to be involved in the removal of u r a c i l residues in DNA has been p u r i f i e d from e x t r a c t s of E_. col i and human f i b r o b l a s t s . I t i s c a l l e d u r a c i l g l y c o s i d a s e (13,14). It can be concluded from the above argument t h a t d e p u r i n a t i o n / depyrimidination of DNA occurs to a s i g n i f i c a n t extent in v i v o . Considering spontaneous depurination alone, a growing mammalian c e l l may loose 2,000-10,000 purines and a few hundred p y r i m i d i n e residues from i t s DNA during a c e l l generation time of 20 hours (1). 2. P o s s i b l e c e l l u l a r e f f e c t s of a p u r i n i c / a p y r i m i d i n i c s i t e s : Besides a d i r e c t loss of g e n e t i c information, the presence of a p u r i n i c / a p y r i m i d i n i c s i t e s on the DNA has several other conse-quences. During r e p l i c a t i o n , the p o s i t i o n opposite to the a p u r i n i c / a p y r i m i d i n i c s i t e s in the newly formed complementary stran d may be f i l l e d at random. Or, the r e p l i c a t i o n mechanism may simply s k i p the l e s i o n s and d e l e t i o n s res u11. In v i t ro, the f i d e l i t y of DNA s y n t h e s i s by AMV DNA polymerase was found t o decrease with a depu-r i n a t e d poly d(A-T) template (15). A p u r i n i c / a p y r i m i d i n i c s i t e s a l s o lead to chain.breakages (16), i n t e r s t r a n d c r o s s l i n k formation (17) in the DNA, and a d e s t a b i I i z a t i o n of the DNA double h e l i x (18). In T7 c o l i p h a g e , one out of seven t o e i g h t depurination events was reported t o be an i n a c t i v a t i o n h i t (19,20). One can t h e r e f o r e envisage t h a t a p u r i n i c / a p y r i m i d i n i c s i t e s , i f 3. •unrepaired, w i l l impose mutagenic and t o x i c e f f e c t s on a c e l l . DNA r e p a i r mechanisms must have evolved t o safeguard the c e l l from depurination and depyrimi di n a t i o n . ;. 3. DNA r e p a i r mechanisms : Several DNA r e p a i r mechanisms have been proposed t o f u n c t i o n in both procaryotes and eucaryotes f o r the r e p a i r of various DNA l e s i o n s (21-22). They are summarised in Figures 1 and 2. Most of the pathways i l l u s t r a t e d are e x a m p l i f i e d by the r e p a i r of p y r i -midine dimers, from which our present concepts of t h e : v a r i o u s DNA r e p a i r mechanisms are l a r g e l y derived. The formation of t h i s DNA l e s i o n i n v o l v e s a covalent l i n k i n g of adjacent pyrimidines in a DNA strand and i s induced by u l t r a v i o l e t l i g h t . The s i m p l e s t mode of DNA r e p a i r i s a d i r e c t r e v e r s i o n of the damaged DNA back t o the undamaged form. To date, the only well e s t a b l i s h e d example i s the enzymatic p h o t o r e a c t i v a t ion of p y r i m i -dine dimers. In t h i s process, an enzyme c a l l e d photolyase o r p h o t o r e a c t i v a t i n g enzyme i s able t o monomerize the UV-induced p y r i -midine dimers in the presence of l i g h t with a wavelength of 320-370 nm. Recently, i t was suggested t h a t 0^-methyIguanine of a l k y -lated DNA could a l s o be reverted back d i r e c t l y t o the undamaged form v i a an enzyme-mediated deaIky I at ion process. Thus, a s p e c i f i c enzyme c a l l e d demethylase was i s o l a t e d from r a t l i v e r which removed the 0^-methyl group from the a l t e r e d guanine base (23). In the case of a p u r i n i c / a p y r i m i d i n i c DNA, r e p a i r can be accomplished by i n s e r t a s e a c t i v i t i e s which simply place c o r r e c t bases back i n t o l.qKt hi? Photoreaction E x c i s i o n r e p a i r Post-rep I i c a t i o n recombination d imer bypass r e p l i c a t i o n Branch m i g r a t i o n bypass rep I i c a t i o n photo lyase • b i n d i n g UV "endonuclease i nci s ion norma I • rep I i c a t i o n norma I 'rep I i c a t i o n norma I rep Ii c a t i o n X / ^ ;hu absorption spI i t s dimer l„ yi>' exc i s i on & - v \ repa i r rep Ii cat ion recombination error-prone bypass rep I i c a t i o n branch migration & bypass rep I i c a t i o n S*' norma I rep I i c a t i o n normal rep I i c a t i o n r e p a i r rep I i c a t i o n norma I rep I i c a t i o n norma I rep I I c a t i o n parental DNA — -- normal r e p l i c a t i o n •••• r e p a i r or bypass r e p l i c a t i o n A p y r i m i d i n e dimer Figure 1. Schematic representation of the p r i n c i p l e mechanisms f o r the r e p a i r of i n t r a s t r a n d p y r imidine dimers in DNA. For d e t a i l s , see references c i t e d in t e x t . 5. A C G C T A G 5' J^RJ^RJ^RJ-RJ-RJ-PJ-P 3" I c y t o s i n e d e a m i n a t i o n i A C G U T A G 5 - - - ^ j N = J s = J ^ p J v p J . p J . p . 3' u r a c i l g l y c o s i d a s e A C G T A G S L - - ^ P ^ P J - P J - P J ^ P J -P 3 ' a p u r i n i c e n d o n u c l e a s e I A C G T A G P 3 ' 5 ' - — J ^ P J M D H PJ - P^PJP^! e x c i s i o n , r e p a i r r e p l i c a t i o n I A C G C T A G 5 - — v h p v f ^ RJ-PJ-PJ* PJ-P_ _ _ - 3 ' Figure 2. Base e x c i s i o n DNA r e p a i r mechanism f o r the r e p a i r of p a r t l y deaminated DNA. The r e p a i r process takes place on double stranded DNA, but the complementary strand has been omitted f o r s i m p l i c i t y . 6. the l e s i o n , s i t e s . Insertase a c t i v i t y for a p u r i n i c DNA has been • found in human f i b r o b l a s t s (24,25). Another mode of DNA r e p a i r i nvo I ves -:the e x c i s i o n of the dama-ged bases o r n u c l e o t i d e s from the DNA and i s thus termed e x c i -sion r e p a i r . As shown in Figure 1, r e p a i r of pyrimidine dimer i s i n i t i a t e d by a s o - c a l l e d UV-endonuclease which i n c i s e s the DNA ad-j a c e n t t o the l e s i o n . The damaged nuc l e o t i d e s and adjacent nucleo-t i d e s are then excised by an exonuclease a c t i v i t y . F i n a l l y , the gap created during the e x c i s i o n step i s f i l led and sealed by the conj- •• c e r t e d a c t i o n of DNA polymerase and Tigase.- This• i s . c a l j e d " c I a s s -i c a l n u c l e o t i d e e x c i s i o n r e p a i r " . A simi l a r mechanism i s thought t o operate f o r the r e p a i r of a p u r i n i c / a p y r i m i d i n i c s i t e s . A s p e c i f i c endonuclease f o r these DNA l e s i o n s has been p u r i f i e d and characte-r i s e d from sources as divergent as E_. co I i (26,27), c a l f thymus (28), c a l f l i v e r (29), human placenta (30) and p l a n t embryo of Phaseolus m u l t i f l o r u s (31). The enzyme i s g e n e r a l l y known as apu-r i n i c endonuclease. So f a r , no separate endonuclease a c t i v i t i e s have been found f o r a p u r i n i c or a p y r i m i d i n i c s i t e s . In v i t r o , r e p a i r of a p u r i n i c s i t e s has been demonstrated by incubating depurinated DNA with a p u r i n i c endonuclease, DNA polymerase i , the four deoxyribo-nucleoside t r i p h o s p h a t e s , p o l y n u c l e o t i d e Iigase and i t s coenzyme (32). The r e p a i r r o l e of a p u r i n i c endonuclease i s f u r t h e r supported by mutants of JE. c o l ? d e f e c t i v e in t h i s enzyme a c t i v i t y . . These' .mutants are more s e n s i t i v e t o methyl methanesuIphonate (an a l k y l a t i n g agent) than the w i l d type (33,34). Recently, a base e x c i s i o n r e p a i r mode has been proposed. In t h i s 7. p r o c e s s , the f i r s t step i s the removal of the damaged base by an •N-gIycosfdase w h i l e the backbone of the DNA strand remains i n t a c t . The r e s u l t i n g a p u r i n i c or a p y r i m i d i n i c s i t e i s then removed as de-s c r i b e d e a r l i e r . T h i s kind of DNA r e p a i r i s b e l i e v e d to' be respon-s i b l e f a r the removal of u r a c i l o r a l k y l a t e d bases from the DNA molecule (35,36) ( F i g . 2). The remaining DNA r e p a i r pathways can be grouped together in a c l a s s termed daughter strand r e p a i r (25). With t h i s mode of DNA r e p a i r the l e s i o n s - a r e not- removed from- the DNA, but are merely d i l u t e d out as a r e s u l t of DNA r e p l i c a t i o n . These r e p a i r mechanisms are by f a r the l e a s t understood in term of the enzymes o r p r o t e i n s i n -volved. In the process of p o s t r e p I i c a t ion recombination, the normal r e p l i c a t i o n mechanism apparently bypasses the damage and leaves a gap i n the daughter stra n d opposite the damaged region. This gap i s then f i l l e d v i a a recombination event with the undamaged parental! DNA as i l l u s t r a t e d in Figure 1. This r e p a i r process i s error-prone. Another error-prone r e p a i r model c a l l e d bypass re-p l i c a t i o n has been proposed a few years ago (37). According t o t h i s model, the p o s i t i o n opposite the l e s i o n in the newly formed complementary strand i s f i l l e d at random. There i s a l s o an e r r o r - f r e e bypass r e p l i c a t i o n (38). This process involves a branch m i g r a t i o n in which the two daughter strands anneal t o one another. Then the gap corresponding t o the l e s i o n s i t e in one daughter s t r a n d fs f i l l e d by using the other daughter stra n d as a template. 8. 4. A p u r i n i c endonuclease in human c e l l l i n e s : Several' human g e n e t i c diseases are a s s o c i a t e d with a DNA re-p a i r d e f i c i e n c y (39-41). A p u r i n i c endonuclease a c t i v i t y has been analysed in crude e x t r a c t s of f i b r o b l a s t s derived from p a t i e n t s with these diseases, and i s w i t h i n the normal range in the cases of a t a x i a t e l a n g i e c t a s i a , Fanconi's anemia, Bloom's syndrome, Cockayne's syndrome and pro g e r i a (42,43). However, in some c e l l l i n e s of xeroderma pigmentosum, a p u r i n i c endonuclease a c t i v i t y was shown t o be d e f e c t i v e (44,45). 5. Inherited DNA r e p a i r defects in xeroderma pigmentosum : Xeroderma pigmentosum (XP) i s an autosomal r e c e s s i v e d i s e a s e , p a t i e n t s are extremely s e n s i t i v e t o s u n l i g h t and have a high incidence of s k i n cancer. When XP c e l l s were t r e a t e d with UV l i g h t o r chemicals such as 4 - n i t r o q u i n o l i n e - 1 - o x i d e , bromobenz(a)-anthracene o r acetyI aminoffuorene, it-was found t h a t e x c i s i o n r e p a i r was d e f i c i e n t in most XP c e l l l i n e s , but normal in others (46-48). The l a t t e r group of XP c e l l s i s c a l l e d XP v a r i a n t s . Using Sendai v i r u s , f i b r o b l a s t s from d i f f e r e n t XP c e l l l i n e s can be fused. The r e s u l t i n g heterokaryons may or may not have a normal le v e l of DNA r e p a i r s y n t h e s i s to UV damage. These experiments led t o the assignment of various e x c i s i o n r e p a i r - d e f i c i e n t XP c e l l l i n e s i n t o f i v e compIementation groups: They are designated as groups A, B, C, D, E (49). Recently, the p o s s i b l e e x i s t e n c e of three more complementation groups has been suggested (47). XP v a r i a n t s wiI complement with a l l other c e l l s t r a i n s . • •-I t i s . g e n e r a l l y agreed t h a t XP group A-E c e l l s have a defect in the i n o i s i o n step of the n u c l e o t i d e e x c i s i o n r e p a i r pathway f o r p y r imidine dimers (46,47), and c e l l s of XP v a r i a n t s are defec-t i v e i n postrep I i c a t i o n r e p a i r (50). Recent data i n d i c a t e they may be d e f e c t i v e in other modes of DNA r e p a i r as w e l l . Thus, XP c e l were found t o have a lower leve l of p h o t o r e a c t i v a t i n g enzyme than normal c e l l s (51,52). A p a r t i a l defect in p o s t r e p I i c a t i o n re-p a i r was a l s o observed in XP group A-D c e l l s (53). Kuhnlein et aj_. (44) reported t h a t f i b r o b l a s t s from XP group D c e l l l i n e s had about one - s i x t h of the normal a p u r i n i c endonu-? clease a c t i v i t y . The apparent Michael i s constants (K^) of the a p u r i n i c endonuclease a c t i v i t y in e x t r a c t s from XP group A and D c e l l s were higher than those of normal c e l l s . I n t e r e s t i n g l y , only p a t i e n t s of XP group A and D show n e u r o l o g i c a l complications (54). Whether abnormal a p u r i n i c endonuclease a c t i v i t y has any e t i o l o g i c a l r o l e in the n e u r o l o g i c a l symptoms in the XP p a t i e n t s remains a question. A p u r i n i c endonuclease a c t i v i t y in the crude e x t r a c t s of normal human f i b r o b l a s t s was resolved by phosphoceI IuIose co-lumn chromatography i n t o two species : an a c t i v i t y (flow-through a c t i v i t y ) t h a t d i d not adsorb t o the column at 10 mM KPO^ concen-t r a t i o n and another a c t i v i t y ( h i g h - s a l t e l u a t e ) t h a t e l u t e d from the column at about 240 mM KPO^ c o n c e n t r a t i o n . The flow-through a c t i v i t y had a higher sedimentation c o e f f i c i e n t of 3.3 S and an apparent of 5 nM a p u r i n i c s i t e s , w h i l e the corresponding values f o r h i g h - s a l t e l u a t e a c t i v i t y were 2.8 S and 44 nM r e s p e c t i v e l y (45) 10. Flow-through a c t i v i t y was not detected i n f i b r o b l a s t e x t r a c t s from XP-D c e l l l i n e s . In f i b r o b l a s t s of XP v a r i a n t s , the level of u r a c i l • DNA N-glycosidase a c t i v i t y was claimed t o be roughly h a l f of t h a t of normal f i b r o b l a s t s (55). No abnormality was revealed in other XP c e l l s . These observations suggest the base e x c i s i o n r e p a i r me-chanism i s d e f e c t i v e i n at l e a s t some XP c e l l s . Thus, when c e l l s from an XP group A c e l l l i n e were exposed t o an alkylating./agent, such as e t h y l n i t r o s o u r e a , the frequency of s i s t e r chromatid exchanges was s e v e r a l f o l d higher than t h a t of normal c e l l s s i m i l a r l y t r e a t e d (56). A slower removal ra t e f o r 0^-aIkyIguanine was a l s o reported in t h i s XP group A c e l l l i n e (57). 11. 6. O b j e c t i v e s : From the above d i s c u s s i o n , one can i n f e r t h a t whatever the primary biochemical defect in XP c e l l s i s , i t has a p l e i o t r o p i c e f f e c t on d i f f e r e n t DNA r e p a i r mechanisms. One p o s s i b i I i f y t o ex-p l a i n t he g e n e t i c heterogeneity and m u l t i p l e enzymatic d e f i c i e n c i e s of XP i s t h a t the d i f f e r e n t r e p a i r enzymes share common regulatory components. One o r more of these components may be d e f e c t i v e or pro-duced i n reduced l e v e l s in XP c e l l s . There i s as yet no documen-t a t e d evidence f o r the e x i s t e n c e of r e p a i r enzyme complexes except perhaps the c o n t r o v e r s i a l endonuclease II a c t i v i t y f rom E. ICQ I i ( 6 ) . This enzyme co n t a i n s a p u r i n i c endonuclease a c t i v i t y and i s a l s o capable of r e l e a s i n g O^-mefhyIguanine and 3-methyIadenine from me-t h y l a t e d DNA. Endonuclease II may thus be a preparation c o n t a i n i n g s e v e r a l d i f f e r e n t enzymes (6,1, 26, 34, 35). A s t a b l e dimer of "endonuclease I I " a n d a p u r i n i c endonuclease can a l s o be formed (58). An i n t e r e s t i n g phenomenon was revealed when flow-through a p u r i n i c endonuclease a c t i v i t y from human f i b r o b l a s t s was re-a p p l i e d t o the phosphoceI Iulose column. The column was e l u t e d f i r s t with a 10 BM and then a 0.3 M KP0 4 b u f f e r . I t was r e -ported t h a t about 80$ of the recovered a c t i v i t y was again found in the fiow-through f r a c t i o n s w h i le the remaining a c t i v i t y now only e l u t e d w i t h 0.3 M KPO^ b u f f e r . This r e s u l t suggests a pos-s i b l e c o n v e r s i o n o f the flow-through enzyme species t o the high-s a l t e l u a t e form. As discussed e a r l i e r , the flow-through enzyme species has a lower and p o s s i b l y a higher molecular weight than the h i g h - s a ! t e l u a t e s p e c i e s . An accessory molecule may 12. complex with the h i g h - s a l t e l u a t e a p u r i n i c endonuclease t o form the flow-through s p e c i e s . To pursue t h i s problem f u r t h e r , a large amount of c e l I e x t r a c t i s needed. An a l t e r n a t i v e source of mate-r i a l t h e r e f o r e seems more p r e f e r a b l e than human f i b r o b l a s t s . A p o s s i b l e candidate i s a human c e l l l i n e c a l l e d Hela c e l l s , c e l l s of which can grow in suspension c u l t u r e . Before going i n t o any large s c a l e study, i t i s necessary t o e s t a b l i s h i f Hela c e l l s have a l s o a flow-through and a h i g h - s a l t e l u a t e species of a p u r i n i c endonuclease as human f i b r o b l a s t s . In t h i s t h e s i s , I s h a l l r e p o r t the p a r t i a l p u r i f i c a t i o n and c h a r a c t e r i s a t i o n of a p u r i n i c endonucl-eases from Hela c e l l s . The p u r i f i c a t i o n method was according t o Kuhn l e i n et aj_. (45). The a n a l y s i s of a p u r i n i c endonuclease a c t i v i t y from Hela c e l l s in i t s e l f i s i n t e r e s t i n g s i n c e t h i s c e l l l i n e i s n e o p l a s t i c in o r i g i n (59). Research in the past decade has i n d i c a t e d a r e l a t i o n -s h i p between p r o f i c i e n c y of DNA r e p a i r mechanism and s u s c e p t i b i l i t y of an i n d i v i d u a l t o cancer (39,60). Crude e x t r a c t from Hela c e l l s was reported to have a higher endonuclease a c t i v i t y f o r U V - i r r a -d i a t e d DNA (61), yet i t s a p u r i n i c endonuclease a c t i v i t y was s i m i l a r t o those of normal human f i b r o b l a s t s (42). I t was pointed out t h a t in v i t r o measurement of composite a c t i v i t y of m u l t i p l e endonucleases fn crude e x t r a c t might not.reveal a d e f i c i e n c y of the act u a l r e p a i r enzyme.' An example was the f i n d i n g t h a t i n Fi. c o l i UV endonuclease a c t i v i t y was the same i n crude e x t r a c t s of w i l d type and UV-dimer e x c i s i o n - d e f i c i e n t mutants. The paradox was re-solved by the demonstration of two UV endonucleases, one of which 1 3 . was absent in the mutants (62). Therefore, one of my o b j e c t i v e s was t o see i f I could show any d i f f e r e n c e in a p u r i n i c endonuclease a c t i v i t y between Hela c e l l s and human f i b r o b l a s t s . The d i f f e r e n t species of a p u r i n i c endonuclease from Hela c e l l s were c h a r a c t e r i s e d with respect t o t h e i r optimal requirements f o r MgCI 2 > pH optimums, heat s e n s i t i v i t i e s , s a l t con-c e n t r a t i o n dependences and molecular weights. To provide f u r t h e r evidence f o r a conversion of the flow-through species t o the h i g h - s a l t e l u a t e species of a p u r i n i c endonuclease, the p r e l i m i n a r y experiment of of phosphocelIulose column rechromatography as.described e a r l i e r in t h i s s e c t i o n , was repeated in more d e t a i l s . A l i n e a r g r a d i e n t of KP0 4 s o l u t i o n was used as e l u t i o n b u f f e r in place of 300 mM KP0 4 s o l u t i o n . The o b j e c t i v e was to show t h a t enzyme a c t i v i t y derived from the flow-through species of a p u r i n i c endonuclease was a l s o e l u t e d from the phosphocelIulose column at around 240 mM KPO. c o n c e n t r a t i o n . 14. MATERIALS AND METHODS 1. Tissue c u l t u r e : i ^ Ce I I Ii nes : Hela c e l l s were a g i f t from Dr. J.B. Hudson of Micro-biology Department, U n i v e r s i t y of B r i t i s h Columbia. Peggy c e l l s , were human f i b r o b l a s t s grown from a s k i n punch biopsy from a normal Cau-casian female. (b) C u l t u r e media : Dulbecco's modified Eagle's medium (Gibco) was r o u t i n e l y supplemented with 10$ of f e t a l c a l f serum (Gibco) and the f o l l o w i n g a n t i b i o t i c s : P e n i c i l l i n (80 u n i t s / m l , f i n a l concen-t r a t i o n ) , streptomycin sulphate (23.7 pg/ml), Kanamycin UOOyg/ml) and Fungizone (2.5 ug/ml). The a n t i b i o t i c s were a l l purchased from Gibco. The medium was adjusted t o pH 7.0-7.5 with 7.5$ sodium bicarbonate s o l u t i o n . The c u l t u r e medium was s t e r i l i z e d by f i l t e r i n g through a S a r t o r i u s membrane paper with a pore s i z e of 0.2 ym. ^ CeI I growth : C e l l s were grown in 32 ounce p r e s c r i p t i o n b o t t l e s (Brockway Glass Co. Inc.) with 50 ml of cu I t u r e media. Incubation was a t 37°C ;n a humidified incubator with 5% C 0 2 and 95$ a i r . Confluent c e l l s were s p l i t 1:4 a f t e r treatment with t r y p s i n s o l u -t i o n (Gibco). (d) CelI h a r v e s t i ng : C e l l s were harvested in batches of 6-12 bot-t l e s when they were near confluency. The c e l l c u l t u r e media were poured o f f . C e l l s were washed twice with 10 ml of phosphate-buffered s a l i n e (25 mM KP0 4 ( pH 7.0V0.15 M NaC!/0.015M sodium c i t r a t e ) and suspended in 40 ml of phosphate b u f f e r s a l i n e by sc r a -ping, from the b o t t l e s u r f a c e . A l t e r n a t i v e l y , the c e l l s were r e -15. leased from the b o t t l e s urface a f t e r incubation w i t h 5 ml of t r y p s i n ' a t 37°C f o r 10 min. The c e l l s were then washed twice in phosphate ! b u f f e r s a l i n e b y c e n t r i f u g a t i o n and resuspension. C e l l p e l l e t s w e r e h s t o r e d e i t h e r d i r e c t l y i n l i q u i d n i t r o g e n o r they were f i r s t resuspend in 2 mi of 50 mM Tris-HCl(pH 7,5) before storage. 2. P r e p a r a t i o n of PM2 phage DNA : Methods f o r the c u l t u r i n g s of Pseudomonas Bal~31 b a c t e r i a and PM2 phage were modified from those of Espejo and Cane Io (63). (a) B a i - b r o t h : I I , of s t e r i I i zed s o l u t i o n contai ned 10 mM T r i s -HCl (pH 7.5), 12 gm of magnesium sulphate (MgS0 4.7H20, Sigma), 26 gm of sodium c h l o r i d e ( F i s h e r ) , 8 gm of b a c t o n u t r i e n t broth ( D i f c o ) , 0.01 M of calcium c h l o r i d e , 3.5 ml of 20% potassium c h l o r i d e . (b) Bal-top agar : 5 gm of bacto~agar ( d i f c o ) was d i s s o l v e d in I I . of B a l - b r o t h and s t e r l i z e d by a u t o c l a v i n g . I t was l i q u i f i e d by heating in a water bath a t 50°C before use. (c) Bal-pI ate : 23 gm of bacto-agar (Difco) was d i s s o l v e d in 1 1 . of B a l - b r o t h and s t e r l i z e d by a u t o c l a v i n g , The agar s o l u t i o n was d e l i v e r e d i n a l i q u o t s on p l a s t i c dishes (87.5 mm x 15 mm Can l a b ) , and allowed t o s o l i d i f y at room temperature. (d) Plaque assay f o r phage t i t e r : Pseudomonas BaI-31 b a c t e r i a were grown in an aerated t e s t tube with about 10 ml Bal-broth a t 28°C ov e r n i g h t . For phage assay, 3 ml of Bal-top agar was added t o 0.1 ml of overnight b a c t e r i a c u l t u r e with 0.1 ml of phage a l i q u o t . The s o l u t i o n was mixed and poured on the B a I - p l a t e s and incubated o v e r n i g h t a t room temperature. The phage t i t e r was 16. estimated by counting the number of plaques appeared. ' (e) P r e p a r a t i o n of phage stock : Pseudomonas BaI-31 b a c t e r i a were grown tn 200 t o 500 ml of Bal- b r o t h a t ,28°C t o a density of 2 x 10^/ml and i n f e c t e d with PM2 phage a t an MOI of 10"? phage per bact-erium. The c u l t u r e was incubated overnight. The b a c t e r i a and c e l l d e b r i s were p e l l e t e d by c e n t r i f u g a t i o n a t 10,000 rpm f o r 15 min with a Beckman Type 21 r o t o r . The suspernatant was used as the phage stock and u s u a l l y had a t i t e r of 5 x 10^/ml. ( f ) P r e p a r a t i o n of 3 H - l a b e l l e d PM2 DNA : B a c t e r i a Pseudomonas B a l -31 were grown i n a 1.5 I. Bal-broth in a 3 I. erlenemeyer f l a s k a t 28 WC. Aerat ion was created by s t i r r i n g the c u l t u r e v i g o r o u s l y with 8 a magnetic s t i r r e r . When the b a c t e r i a reached a density of 3 x 10 /ml ( t t t e r e d w i t h a Petroff-Houser b a c t e r i a counter), 0.15 gm of deoxyadenine (Sigma) was added t o the medium. Five min l a t e r , the 12 c e i l s were i n f e c t e d w i t h 1^2 x 10 PM2 v i r u s . A f t e r 5 min, 1.5 mCi of methyi-^H thymidine ( s p e c i f i c a c t i v i t y 50 Ci/mmole, New England Nuclear) was added and the c u l t u r e was incubated overnight. Bac-t e r i a and c e l l d e b r i s were removed by c e n t r i f u g a t i o n f o r 15 min-at 10,000 rpm w i t h a Beckman Type 21 r o t o r . The supernatant was c e n t r i f u g e d f o r 3 hours at 20,000 rpm with the same r o t o r . The p e l l e t which contained the phage p a r t i c l e s was resuspended i n about 15 ml of RB b u f f e r c o n t a i n i n g 20 mM Tri s - H C l (pH 8.0)/1 M NaCl and c e n t r i f u g e d f o r 10,000rpm f o r 15 min in a Beckman Type 50 Ti r o t o r . The p e l l e t was discarded, the supernatant was c e n t r i -fuged f o r 50 min.at 50,000 rpm in the same r o t o r . The pelletvwas resuspended in 15 ml of RB b u f f e r and c e n t r i f u g e d f o r another 17. 10,000 rpm f o r 15 min. The f i n a l supernatant was made t o have a •density of about 1.28 gm/cc with C s C l . The phage p a r t i c l e s were i. banded by c e n t r i f u g a t i o n in a polyallomer tube with a Beckman Type ;. 50 T i r o t o r a t 40,000 rpm f o r 24 hours at 20°C. A f t e r the CsCl density g r a d i e n t c e n t r i f u g a t i o n , one major band of phage p a r t i c l e s was evident in the middle of the polyallomer tube ( F i g . 3 ) . A minor band of m a t e r i a l was a l s o evident below the major band. The ma t e r i a l in t h i s minor band was not analysed and was discarded. The m a t e r i a l in the major band was c o l l e c t e d and d i a l y s e d against 1 I. of 0.02 M Tri s - H C l (pH 7.5)/0.1 M NaCI/ 1 mM EDTA f o r 3 hours or more. The phage was lysed by 10$ SDS added dropwise u n t i l the s o l u t i o n was c l e a r e d . The phage DNA was e x t r a c t e d in the aqueous phase by phenol e x t r a c t i o n as described by Espejo et aj_. (64). The PM2 DNA was then d i a l y s e d e x t e n s i v e l y against 0.01 M T r i s (pH 7.5). PM2 DNA concentration was determined by measuring the absor-TM bance at 260 nm. The molar e x t i n c t i o n c o e f f i c i e n t E. 260 nm of 1 cm PM2 DNA was assumed t o be 6.5 x 10 3. A t y p i c a l r e s u l t where the PM2 DNA had been d i l u t e d 10-fold and gave an °-D-260nm o f 0 , 3 2 -was as shown in Figure 4. The co n c e n t r a t i o n of the DNA was c a l c u l a t e d as 0.49 mM. Typical y i e l d s f o r a 1.5 I. c u l t u r e were 10-15 umoles n u c l e o t i d e of DNA with a r a d i o a c t i v i t y of 6,000-8,000 cpm/nmole. 3. Enzyme p u r i f i c a t i o n : A l l p u r i f i c a t i o n procedures were c a r r i e d out at 2°C. (a) P r e p a r a t i o n of c e l l e x t r a c t : Frozen c e l l s were thawed and resus-18. Figure 3. I s o l a t i o n of PM2 phage p a r t i c l e s by cesium c h l o r i d e d e n s i t y - g r a d i e n t e q u i l i b r i u m c e n t r i f u g a t i o n . The upper w h i t i s h band contained the phage p a r t i c l e s (arrowed). 19. Figure 4. U l t r a v i o l e t absorption spectrum of p u r i f i e d PM2 phage DNA in 10 mM T r i s - H C l (pH 7.5) PM2 DNA 10 mM T r i s - H C l (pH 7.5) 20. pended in 2 ml of 50 mM Tris - H C l (pH 7.5)70.1 mM DTT. The c e l l s were disrupted with sonic i r r a d i a t i o n 6 times f o r 15 sec each. 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 at 50,000 rpm in a Beck-man Type 50 Ti r o t o r and the p e l l e t discarded. The supernatant f l u i d (high speed supernatant) was subjected t o f u r t h e r p u r i f i c a -t i o n . 10 b o t t l e s normally y i e l d e d 8-10 mg of s o l u b l e p r o t e i n . (b) DEAE-celIulose chromatography : A column of Whatman DE-22 DEAE-cel I ulose (5 mm x 35 mm) was prepared and e q u i l i b r a t e d with b u f f e r A (50mmM Tris-HCl (pH 7.5)/0.4 M NaCI/10$ g l y c e r o l / 0 . 1 mM DTT). The high speed supernatant was adjusted.to have the s a m e b u f f e r content as b u f f e r A and loaded onto the column at a flow r a t e of 0.125 ml/min. The column was then washed with b u f f e r A at the same flow r a t e . F r a c t i o n s of 1 ml were c o l l e c t e d , u s u a l l y the f i r s t four f r a c t i o n s c o n t a i n i n g most of the a c t i v i t y were pooled and d i a l y s e d overnight against two 400 ml a l i q u o t s of b u f f e r B c o n t a i n i n g 10 mM KP0 4(pH 7.4)/ 10$ g l y c e r o l / 0 . 1 mM DTT. The f i n a l d i a l y s a t e (DEAE pool) was re-t a i n e d . (c) PhosphoceI Iulose chromatography : A column of Whatman P-11 phosphoceI IuIose (1.1 cm x 3.5 cm) was prepared and e q u i l i b r a t e d by washing with b u f f e r B. The DEAE pool was a p p l i e d t o the column at a flow rate of 0.04 ml/min. The column was then e l u t e d with 7-to 9 ml of b u f f e r B, then 5-to 5 ml of 50 mM KP0 4(pH 7.4)/10$ glyce r o l / 0 . 1 mM DTT and a 46 ml l i n e a r g r a d i e n t of 50 t o 400 mM „KP0 4(pH 7.4)/10$.glycerol/0.1 mM DTT. F r a c t i o n s of 1 ml were c o l -l e c t e d in p l a s t i c tubes. 10 uI of 10 mg/ml a c e t y l a t e d BSA was added t o each f r a c t i o n t o s t a b i l i z e the enzyme a c t i v i t y . The f r a c t i o n s 21. with most enzyme a c t i v i t y were made t o 35$ g l y c e r o l and s t o r e d at / -20°C. (4) PM2 DNA depurination : Depurination b u f f e r was made up of 1 M NaCI, 0.1 M sodium c i -t r a t e and adjusted to pH 4.0 with HCI. The depurination b u f f e r was then d i l u t e d 10-fold with PM2 DNA which was in 10 mM Tris-HCl (pH 7.5). Thus, depurination was c a r r i e d out at 70°C f o r 15 min with 0.5 mM DNA in 9 mM T r i s - H C l , 0.1 M NaCI, and 0.01 M sodium c i t r a t e . The f i n a l pH of the s o l u t i o n was 4.6. (5) F i l t e r - b i n d i n g assay : 0.15 ml of 0.01$ SDS/0.25 mM EDTA (pH 7.0) was added t o the DNA r e a c t i o n mixture followed by 0.2 ml of 0.3 M K 2HP0 4*K0H (pH 12.4). A f t e r 2 min at room temperature, the s o l u t i o n was n e u t r a l i z e d with 0.1 ml of 1 M KH 2P0 4-HCI (pH 4.0). This treatment was found t o denature nicked PM2 DNA, but not c o v a l e n t l y closed molecules. However, when the pH of the 0.3 M K 2HP0 4-K0H added was above pH 12.8, unnicked PM2 DNA a l s o became denatured ( F i g . 5). 0.2 ml of 5 M NaCI and 5 ml 50 mM Tri s - H C l (pH 8.01/1 M NaCI were then added s u c c e s s i v e l y . The s o l u t i o n 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 ( S c h l e i c h e r and Schnell type BA 85, 0.45 ym pore s i z e ) which s e l e c t i v e l y r e tained denatured DNA (65). The f i l -t e r was washed with 5 ml of 0.3 M NaCI/0.03 M sodium c i t r a t e , d r i e d and counted in a l i q u i d s c i n t i l l a t i o n c o u n t e r . ( S e a r l e , D e l t a i 3 0 0 , l i q u i d s c i n t i I I at ion system) with 5 ml of s c i n t i I I at ion f l u i d . 22. too •* 80 0 oO 40 20; pH 13.1 + 10 pi 5N KOH 118 12.2 p H 12.6 13.0 Figure 5. S t a n d a r d i z a t i o n of f i l t e r - b i n d i n g assay. The f i l t e r - b i n d i n g assay was c a r r i e d out as described in M a t e r i a l s and Methods, except the 0.3 M K 2HP0 4-K0H added was of various pHs as i n d i c a t e d . Untreated DNA was used in t h i s experiment. I t contained less than 0.2 nicks/mo I e c u l e . 23. S c i n t i l l a t i o n f l u i d was made up of 3.8 I. s c i n t i l l a t i o n grade toluene ( F i s h e r ) with 15.2 gm PPO (Syndel Lab. Ltd.) and 0.38..gm f of POPOP (Syndel Lab. L t d . ) . T o t a l DNA presented in a r e a c t i o n mixture was estimated by measuring the r a d i o a c t i v i t y of an a l i q u o t spotted on a blank f i l -t e r-paper. The pro p o r t i o n of nicked PM2 DNA (X) ret a i n e d on the f i l t e r paper was then c a l c u l a t e d . The average number of nicks/, molecule in the DNA (tu) was obtained from the equation a> * - I n ( l - X ) . The equation was derived by assuming a Poisson d i s t r i b u t i o n of the t a r g e t s i t e s among the DNA.mo I ecu Ies (44). Assuming the PM2 DNA had 18,000 n u c l e o t i d e s , the amount of PM2 c i r c l e s in a 50 u l r e a c t i o n c o n t a i n i n g 0.05 mM PM2 DNA nu c l e o t i d e was 138.8 fmoles. Thus, the t o t a l n i c k s in a r e a c t i o n mixture was estimated. 6. A p u r i n i c endonuclease assay : Endonuclease a c t i v i t y was assayed by monitoring the conversion of s u p e r h e l i c a l PM2 DNA t o nicked c i r c l e s . Unless otherwise s t a t e d , a standard r e a c t i o n mixture (0.05 ml) contained 0.05 mM depurinated PM2 3H-DNA n u c l e o t i d e , 50 mM Tris - H C l (pH 8.0), 10 mM MgCI 2, 10 mM KCI, 10 mg/ml of a c e t y l a t e d BSA, a 100-fold d i l u t i o n of the depurin-a t i o n b u f f e r introduced with the depurinated DNA and an appr o p r i a t e amount o f enzyme. A f t e r an incubation f o r 10 min at 37°C, the r e a c t i o n s were c h i l l e d and the f i l t e r - b i n d i n g assay c a r r i e d out as described ear-l i e r . Assays were c o r r e c t e d f o r nicks occuring in the DNA prep a r a t i o n and during d e p u r i n a t i o n . The blank was.0.2-0.4 nicks/molecules. A u n i t of endonuclease a c t i v i t y c a t a l y s e d the production of 24. 1 pmole of nicks per min. Under our c o n d i t i o n s , the assay gave a l i n e a r response with enzyme added up t o a level which produced app-roximately one i n c i s i o n per molecule. The lowest level of n i c k i n g we used t o c a l c u l a t e enzyme a c t i v i t y was at least 10$ g r e a t e r than the blank. 7. Sephadex G-100 column chromatography : A column (0.9 cm x 27.5 cm) of Sephadex G-100 ( p a r t i c l e s i z e 40-120 um,Sigma) was prepared and e q u i l i b r a t e d with two d i f f e r e n t b u f f e r s : b u f f e r X of 50 mM Tri s - H C l (pH 7.5)/50 mM KCI/0.1 mM DTT/ 10$ g l y c e r o l and b u f f e r Y of (50 mM Tri s - H C l (pH 7.55/1 M KCI/0.1 mM DTT/10$ g l y c e r o l . To avoid s t i r r i n g up of the gel during sample loading, a piece of Whatman no.1 f i l t e r paper was placed over the gel s u r f a c e , and a layer of e l u t i o n b u f f e r was put on top of the column. The sample (0.5 ml) was made t o 35$ g l y c e r o l and layered onto the column c a r e f u l l y with a: pasteur p i p e t t e . The column was e l u t e d with b u f f e r A o r B at a rate of 0.056 ml/min. F r a c t i o n s of 0.5 ml were c o l l e c t e d . The f o l l o w i n g standard p r o t e i n s were used f o r c a I i b r a t i o n and were purchased from Sigma : BSA, ovalbumin (egg w h i t e ) , 8-lactoglobuI in A and B ( m i l k ) , myoglobin (whale s k e l e t a l muscle, Type I I ) , c y t o -chrome C (horse heart, Type V I ) . The molecular weights of these p r o t e i n s are 64,000, 45,000, 35,000, 18,000, 12,384,respectively. The void volume (Vo) of the column was measured with blue dextran (average molecular weight 2,000,000, Sigma), the t o t a l volume (Vt) was determined with bromocresol purple (molecular weight 540.2, Sigma). The absorbance at 660 nm was determined f o r each column f r a c t i o n . The e l u t i o n volume (Ve) f o r each standard p r o t e i n was determined by the absorbance at 280 nm. The e l u t i o n constant (Kav) was c a l -c u l a t e d by the equation Kav = (Ve-Vo)/(Vt-Vo). 8. Sucrose g r a d i e n t c e n t r i f u g a t i o n : 4.4 ml of a l i n e a r g r a d i e n t of sucrose (5-20$) was layered above a 0.25 ml cushion of 60$ sucrose in a polyallomer tube (Beck-man). The sucrose s o l u t i o n s used t o form the gr a d i e n t contained 50 mM T r i s - H C l (pH 7.5)/0.1 mM DTT and f o r some experiments, 1 M KCI a l s o . The enzyme sample t o be analysed was f i r s t d i a l y s e d o v e r n i g h t against two changes of 500 ml of 50 mM Tris-HC! (pH 7.5). To minimize disturbance of the g r a d i e n t , 0.25 ml of the enzyme sample was l a -yered on top of the grad i e n t from a microp.ipette attached t o a \ ml s y r i n g e . C e n t r i f u g a t i o n was f o r 27 hours a t 50,000. rpm in a Beckman SW 50.1 r o t o r at 2°C. F r a c t i o n s of 0.2 ml were c o l l e c t e d f o r enzyme assays. BSA (4.25 S ) , 8-lactogIobuIin (2.85 S ) , myoglo-bin (2.0 S) were used as marker p r o t e i n s . 9. S a l t treatment of enzyme a l i q u o t s : The enzyme a l i q u o t s were made t o 2 M KCI or 2 M NaCl and i n -cubated on i c e f o r an hour. They were then subjected t o a n a l y s i s by e i t h e r Sephadex column chromatography o r sucrose g r a d i e n t c e n t r i f u g a t i o n . In some experiment, the a l i q u o t s were-centrifuged in a t a b l e - t o p Eppendorf t a b l e c e n t r i f u g e f o r 5 min. No d i f f e r -ence was observed between the experiments with o r without the 26. c e n t r i f u g a t i o n step. : 10. Preparation of a c e t y l a t e d BSA : 1 gm of BSA (Sigma) was d i s s o l v e d in 25 ml of satura t e d sodium a c e t a t e and 25 ml of 0.2 N sodium phosphate ( d i b a s i c ) and cooled t o 0°C on i c e . While s t i r r i n g on i c e , 50 pi of a c t e t i c anhydride was added t o the BSA s o l u t i o n every 30 min f o r a t o t a l of s i x a d d i t i o n s . The s o l u t i o n was s t i r r e d f o r a f u r t h e r 45 min and then d i a l y s e d e x t e n s i v e l y a g a i n s t d i s t i l l e d water. The s o l u t i o n was n e u t r a l i z e d to pH 7 with 5 N NaOH, and stored at -20°C. This treatment sho Id destroy various contaminating enzyme a c t i v i t i e s as well as g r e a t l y reduce the a f f i n i t y of BSA f o r various small molecules. 11. P r o t e i n determinations : P r o t e i n concentration was measured by the method of Lowry et  a I. (66) using BSA as a standard. 27. RESULTS 1. Q u a n t i f i c a t i o n of'number of a p u r i n i c / a p y r i m i d i n i c s i t e s in de- pur inated DNA : A p u r i n i c s i t e s i n the DNA are s u s c e p t i b l e t o a l k a l i h y d r o l y s i s . The number of a I k a I i - I a b i I e s i t e s in the depurinated DNA i s in good agreement with the number of s i t e s s u s c e p t i b l e t o the a p u r i n i c endonuclease a c t i v i t y (16,45). To achieve a I k a I i h y d r o l y s i s , the f i l t e r - b i n d i n g assay was modified by leaving the r e a c t i o n mixture in a l k a l i c o n d i t i o n f o r a prolonged period of time a t 37°C. A l k a l i h y d r o l y s i s of depurinated DNA was found t o be complete in 40-60 min at 37°C ( F i g . 6). The number of a p u r i n i c s i t e s in the depurinated DNA f o r our enzyme assays was thus estimated t o be about 3.1 n i c k s / molecule ( F i g . 7). 2. P u r i f i c a t i o n of a p u r i n i c endonuclease a c t i v i t y from Hela c e l l s : 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 in Table I. (a) High-speed c e n t r i f u g a t i o n : This p u r i f i c a t i o n step i s necessary t o remove a n o n - s p e c i f i c endonuclease a c t i v i t y (enzyme a c t i v i t y t h a t n i c k s n a t i v e DNA) from the c e l l l y s a t e . This a c t i v i t y was de-t e c t e d in the flow-through f r a c t i o n s ( e l u t e d from the column at 10 mM KPO^ concentration) from the phosphocelIulose column when the c e n t r i f u g a t i o n step was done at a lower speed of 10,000 rpm. The presence of t h i s n o n s p e c i f i c n i c k i n g a c t i v i t y would mask the d e t e c t i o n of an a p u r i n i c endonuclease s p e c i e s . 28. 12 U a o E c ID a U 0.8f c 0.4 / : © — 20 40 60 T i m e , m i n . 80 -//--o--lh 150 Figure 6. Time course of a l k a l i h y d r o l y s i s of a p u r i n i c PM2 DNA. PM2 DNA was depurinated f o r 0 min ( o ) , 3 min (•) and 6 min (•). The DNA was then subjected t o the normal f i l t e r - b i n d i n g assay except t h a t the DNA was l e f t i n the a l k a l i c o n d i t i o n f o r various times a t 37°C. I I I I I I I ! 0 2 4 6 8 10 12 14 d e p u r i n a t i o n t i m e Figure 7. Time course of depurination of FM2 DNA at 70°C. DNA was depurinated f o r various time as i n d i c a t e d and subjected t o the f i l t e r - b i n d i n g assay as described in Figure 6. The DNA was incubated in a l k a l i c o n d i t i o n f o r 45 min a t 37°C before n e u t r a l i z a t i o n . 30. Table I. P u r i f i c a t i o n of a p u r i n i c endonuclease a c t i v i t y from Hela ce I 1S F r a c t i o n Volume P r o t e i n A c t i v i t y S p e c i f i c a c t i v i t y % y i e l d ( m l ) (mg) ( u n i t s ) (units/mg) High speed supernatant 1.7 10.2 8460 830 DEAE pool 3.8 Phospho- I 3 c e l I u l o s e ,. II 4 III 6 6.84 3.3 0.124 0.18 6700 30 80 1650 980 8.2 630 9200 80 0.4 1.0 19.5 31 . (b) DEAE-ce1 Iulose chromatography : This^column was used t o remove n u c l e i c a c i d s from the eel I e x t r a c t (67); About 80-90$ of a p u r i n i c -endonuclease a c t i v i t y and 70-80$ of the t o t a l p r o t e i n was reco-vered from the DEAE-celIulose column. (c) PhosphoceI Iulose chromatography : Three peaks of a p u r i n i c endo-nuclease a c t i v i t y were obtained from the phosphoceI IuIose column. F r a c t i o n s with the most a c t i v i t y were pooled ( F i g . 8) and used in subsequent a n a l y s i s . They are hereby designated as enzyme species Peaks I, II and I I I . Peak I d i d not adsorb t o the phosphocelIuIose column at 10 mM KPO^, Peak II came out from the column a t about 210 mM KP0,, and Peak I II a t 260 mM KPO.. Based on t h e i r a f f i n i t y t o 4 4 ' . phosphoceI IuIose, Peak I and Peak III presumably correspond t o the flow-through and the h i g h - s a l t e l u a t e a p u r i n i c endonuclease a c t i -v i t y of human f i b r o b l a s t s , r e s p e c t i v e l y (45). Peak III was the ma-j o r species of a p u r i n i c endonuclease a c t i v i t y in Hela c e l l s . Peak I and Peak II were r e l a t i v e l y minor s p e c i e s , each amounted t o only 2-5$ the a c t i v i t y of Peak I I I . Peaks I and II did not seem t o be a r t i f a c t s ' r e s u l t i n g from overloading of the column. We obtained s i m i l a r d i s t r i b u t i o n of the three enzyme a c t i v i t i e s in sev e r a l ex-periments where the amount of p r o t e i n put on the phosphoceI IuIose column ranged from 0.47 mg t o 8 mg. Recovery of a p u r i n i c endonuclease a c t i v i t y from the phosphoceI Iu-lose column was g e n e r a l l y about 30$ of the t o t a l a c t i v i t y put onto the column. With the assay c o n d i t i o n s f o r a p u r i n i c endonuclease (see Methods and M a t e r i a l s ) , no s i g n i f i c a n t amount of n o n s p e c i f i c endonuclease 32. F R A C T I O N N O . Figure 8. PhosphocelIulose chromatography of a p u r i n i c endonuclease a c t i v i t y from a Hela c e l l s e x t r a c t . The i n s e r t e d diagram shows the flow-through a c t i v i t y in a l a r g e r s c a l e . a p u r i n i c DNA O O n a t i v e untreated DNA 33. a c t i v i t y ( a c t i v i t y t h a t n i c k s n a t i v e PM2 DNA) was found in the e l u -ate of the phosphoceI IuIose column except in the flow-through f r a c t - f ions. However, when the enzyme assay was performed in a c o n d i t i o n of;' 10 mM T r i s - H C l (pH 7.5), two more peaks of n o n s p e c i f i c endonuclease a c t i v i t y were detected : One e l u t e d from the column a t about 180 mM KPO^ and another a t about 300 mM KPO^. We are now in the progress of determining whether they have any preference f o r other DNA l e -s i o n s . Endonucleases t h a t i n c i s e d n a t i v e DNA were a l s o found in E_. c o I i . Some were shown t o be more a c t i v e on DNA t r e a t e d w i t h UV l i g h t and osmium t e t r o x i d e . One of the endonucleases, endonuclease V, was h i g h l y a c t i v e on u r a c i l - c o n t a i n i n g DNA. This suggested a nu c l e o t i d e e x c i s i o n r e p a i r mechanism f o r removal of u r a c i l residues from the DNA besides a base e x c i s i o n r e p a i r mode u l t i l i z i n g u r a c i I -DNA N-glycosidase (69). Table I a l s o i n d i c a t e s t h a t a t t h i s stage of p u r i f i c a t i o n , Peaks I — tI 1 were s t i l l i n a very crude s t a t e . This was p a r t i c u l a r l y t r u e f o r Peak I, s i n c e the bulk of the p r o t e i n a l s o e l u t e d in the flow-through f r a c t i o n s . The maximum p u r i f i c a t i o n f a c t o r achieved ( c a l -c u l a t e d r e l a t i v e t o the high speed supernatant) was about 10 f o r Peak III. 3. PhosphocelIulose rechromatography of apurinEc endonuclease  a c t i v i t y of Hela c e l l s : Peak I and Peak i l l pools were each d i a l y s e d a g a i n s t two chan-ges of 500 ml of a s o l u t i o n of 10 mM KPO^ (pH 7.4) overnig h t and subjected t o phosphocelIulose chromatography again. 34. When Peak I was rechromatographed, 50-60$ of the recovered a c t i v i t y again e l u t e d in the flow-through f r a c t i o n s . A peak of a c t i v i t y a t around 260 mM KPO^ was a l s o evident. The r e s t of the a c t i v i t y e l u t e d between 170-210 mM KP0 4 ( F i g . 9 ) . In the case of Peak I I I , about 96$ of the recovered a c t i v i t y e l u t e d from the column at 260 mM KP0 4 < However, 4$ of the recovered a c t i v i t y was now recovered i n the flow-through f r a c t i o n s ( F i g . 10). The r e s u l t suggested t h a t f o r a yet undetermined reason, there i s an i n t e r -conversion between the flow-through and the h i g h - s a l t e l u a t e species of a p u r i n i c endonuclease o f Hela c e l l s . To provide f u r t h e r evidence f o r t h i s phenomenon, i t would be of i n t e r e s t t o rechromatograph the flow-through a c t i v i t y f o r a second time on the phosphoceI IuIose column, and t o see i f there w i l l be again a 50 : 50 d i s t r i b u t i o n of enzyme a c t i v i t y in the flow-through and the h i g h - s a l t e l u a t e f r a c -t i o n s . A l a r g e r amount of enzyme e x t r a c t would:be needed for. such experiments. 4. General p r o p e r t i e s of a p u r i n i c endonuclease. a c t i v i t y in Hela c e l l s (a) Requirement of magnesium ions : A l l three enzymes species had some r e s i d u a l a c t i v i t i e s in the absence of d i v a l e n t c a t i o n , and were s t r o n g l y s t i m u l a t e d by the presence of MgC^- They were o p t i m a l l y a c t i v e a t around 5-10mM.MgCl2 ( F i g . 11). MgC^ co n c e n t r a t i o n s above 15 mM were i n h i b i t o r y . (b) pH optimum : Peak I and Peak II had a pH optimum around 8 ( F i g . 12) . In both cases approximately 60 to 70$ of the a c t i v i t y a t op-ti m a l pH was manifested a t pH 7.3 and 8.6. Peak III had an optimum 35. E 1 E A. ?i»->i.-so 3 a 4 Q FRACTION N O F i g u r e 10. P h o s p h o c e I I u I o s e r e c h r o m a t o g r a p h y o f P e a k I • • a p u r i n i c DNA A - A n a t i v e DNA 36. (c) Figure 1 1 . E f f e c t of MgC^ on a p u r i n i c endonuclease a c t i v i t y of He I a ceI Is. (a) Peak I, (b) Peak II and (c) Peak III. Enzyme assays were performed as described in M a t e r i a l s and Methods with the concentrations of MgC^ i n d i c a t e d . A • A a p u r i n i c DNA, A A n a t i v e DNA. 37. cr. P c > P > P 0 • (0 PH 7.5 8.5 optimum ( a ) .A apurinic DNA - A native DNA 20 P c D >* P 15 > 10 65 7.5 8.5 pH optimum 0 0 80 E > 60 P :c 3 >" P 40 > P 0 0 20 6^ PH 7.5 8.5 optimum (c) Figure 12. E f f e c t of pH on a p u r i n i c endonuclease a c t i v i t y of Hela c e l l s , (a) Peak ' I , (b) Peak II and (c) Peak I I I . The f i n a l pH of the standard r e a c t i o n mixture was v a r i e d between 6.1 and 8.6. 38. at around pH 7.5. No s i g n i f i c a n t n o n s p e c i f i c endonuclease a c t i v i t y > i was detected in Peaks II and III over the range of pH t e s t e d . How-l -ever, n o n s p e c i f i c endonuclease a c t i v i t y in Peak I was g r e a t l y stimu-lated at pH below 7 (Fi.g. 12a). A s i m i l a r f i n d i n g was a l s o reported with the human lymphoblastic c e l l l i n e CCRF-CEM (71). (c) E f f e c t s of NaCI and KCI concentration : Increasing c o n c e n t r a t -ions of NaCI seemed t o have an i n h i b i t o r y e f f e c t on a l l three en-zyme species. At 40 mM NaCI, a l l three enzyme a c t i v i t i e s were i n -h i b i t e d t o about 70$ of. the a c t i v i t i e s in the absence of NaCI ( T i g . 13). Peaks I and I I .were a l s o i n h i b i t e d by i n c r e a s i n g concentrations of KCI, while the enzyme a c t i v i t y of Peak III was s l i g h t l y s t i m u l a t e d by the presence of 20-40 mM KCI and was not i n h i b i t e d by KCI concen-t r a t i o n s up t o 100 mMCFig. 14). (d) Heat i n a c t i v a t i o n : A l i q u o t s of Peaks l - l I I were made 50 mM KPO^ (pH 7.4)/10$ glycerol/0.0.1 mM DTT/0.1 mg/m I acety I ated. BSA and heated a t 45°C f o r various time. The r e s u l t s are summarised in Figure 15.. Peak I was q u i t e s t a b l e t o prolonged heating at 45°C, wh i l e the other two species were h e a t - l a b i l e . Peak III was most h e a t - l a b i l e , i t had a h a l f - l i f e of 2 t o 3 min. Enzyme a c t i v i t y in Peak II was i n a c t i v a t e d i n i t i a l I y with a ha If-1 i f e of 2 t o 3 min as Peak IN", the remaining 40-45$ enzyme a c t i v i t y was more heat-st a b l e with a ha I f - l i f e of about 25 min. The r e s u l t suggested the presence of two forms of a p u r i n i c endonuclease in Peak II with markedly d i f -f e r e n t heat s e n s i t i v i t i e s . 5. Molecular weight determinations of Peaks l - l I I : 1 0 0 > > 100 > > o m 5? 1 0 0 > > o SO 0 (a) 4 0 8 0 m M NaCl 4 0 80 m M NaCl (c) 4 0 80 m M NaCl 39. Figure 13. R e l a t i v e a c t i v i t y of (a) Peak I, (b) Peak II and (c) Peak III a t d i f f e r e n t NaCl co n c e n t r a t i o n s . The c o n c e n t r a t i o n of NaCl in the r e a c t i o n mixture was v a r i e d between 0 and 0.1 M. A c t i v i t y at 0-M NaCl was taken as 100$. ( a ) _! I 1 ! 1 40 80 mM KCI 14. R e l a t i v e a c t i v i t y of (a) Peak I, (b) Peak II and (c) Peak III at d i f f e r e n t KCI con c e n t r a t i o n s . The c o n c e n t r a t i o n of KCI i n the r e a c t i o n mixture was v a r i e d between 0 and 0.1 M. A c t i v i t y a t 0 M KCI was taken as. .100$. 41. \ H P e a k H I 1 1 1 _i i 10 25 h e a t i n g t i m e C m i . n . 3 Figure 15. Heat i n a c t i v a t i o n curve of a p u r i n i c endonuclease a c t i v i t y in Peaks l - l I I. A l i q u o t s of Peaks l-lI I were made 50 mM KP0 4 (pH 7.4) by adding 1 M KPO^ in the case of Peak I or by d i l u t i n g with d e s t i l l e d water in the cases of Peaks II and I I I . The a l i q u o t s were heated at 45°C f o r 0-25 min. The r e s i d u a l a p u r i n i c endonuclease a c t i v i t y was determined as described in M a t e r i a l s and Methods. The r e s u l t s , as percentages of i n i t i a l a c t i v i t i e s , were p l o t t e d on a semi-logarithmic s c a l e . 42. A Sephadex G-100 column was used t o 'analyse the molecular weights of the three forms of a p u r i n i c endonuclease a c t i v i t y . I n i -t i a l l y , Sephadex b u f f e r X was used t o e l u t e the enzyme. However, when Peak I of a p a r t i c u l a r experiment was run on the Sephadex G-100 column, most of the recovered a p u r i n i c endonuclease a c t i v i t y e l u t e d in the e x c l u s i o n volume of the Sephadex G-100 column ( F i g . 16). This would imply t h a t the a p u r i n i c endonuclease a c t i v i t y was ass-o c i a t e d with a complex o f . a molecular weight g r e a t e r than 100,000. The remaining a c t i v i t y was d i s t r i b u t e d in the f r a c t i o n s correspond-ing t o molecular weights of 60,000-25,000. S i m i l a r r e s u l t s were obtained with Peaks II and I I I . But in a d d i t i o n , another peak of enzyme a c t i v i t y was obtained in the f r a c t i o n s corresponding t o a molecular weight of 8,000-6,000 ( F i g . 17). When a DEAE pool of Hela c e l l s e x t r a c t was run on the column, the d i s t r i b u t i o n of a p u r i n i c endonuclease a c t i v i t y resembled t h a t of a run of Peak I. These f i n d -ings suggest the "low molecular weight" form of a p u r i n i c endonuclease a c t i v i t y d i s s o c i a t e d from the high molecular weight complex due t o the r e l a t i v e high i o n i c strength (1.2-1.56) of the 200 mM t o 260 mM KP0 4 in the pools of Peaks I I and I I I . We then experimented-with the c o n d i t i o n s t o d i s s o c i a t e a p u r i n i c endonuclease from the high molecular weight complex. For these ex-periments, DEAE pools of Hela eel Is e x t r a c t s were used. I t was found t h a t the high molecular weight complexes d i s s o c i a t e d with i n c r e a s i n g s a l t c o n c e n t r a t i o n s . Incubation of the DEAE pool f o r an hour at 0°C .in 2 M NaCl o r KCI was s u f f i c i e n t to d i s s o c i a t e most of the high molecular weight complex. The a p u r i n i c endonuclease a c t i v i t y would 43. Figure 16. Sephadex G-100 chromatography of Peak I. The column was e l u t e d as described in M a t e r i a l s and Methods with Sephadex e I u t i o n b u f f e r X c o n t a i n i n g 50 mM Tri s - H C l (pH7.5)/50 mM KCI/0.1 mM DTT/10$ g l y c e r o l . B pjapurinic DNA • • n a t i v e DNA 44. Figure 17. Sephadex G-100 chromatography of Peak I I I . The column was el u t e d with Sephadex b u f f e r X. 45. then appear as having a low molecular weight of 6,000-8,000 ( F i g . 18). Since a p u r i n i c endonucleases from otherrsources were reported t o have a monomeric molecular weight of 40,000-28,000 (26-31, 45), our r e s u l t s were unexpected. To check i f these r e s u l t s were p e c u l i a r p r o p e r t i e s of Hela c e l l s o r a r t i f a c t s of the Sephadex column chromatography, the f o l -lowing experiments were devised. We repeated the above experiments with flow-through a c t i v i t y ob-t a i n e d from normal human f i b r o b l a s t s of Peggy c e l l s . A p u r i n i c endo-nuclease was a l s o found t o a s s o c i a t e with a high molecular weight com-plex ( F i g . 19), and a "low molecular weight" form of a p u r i n i c endonu-c l e a s e appeared when the enzyme pool was treated, with 2 M KGI or NaCI. A pool of the "low molecular weight 11 form of Hela a p u r i n i c endonu-clease was then rechromatographed on the Sephadex G-100 column. A smear of a p u r i n i c endonuclease a c t i v i t y was obtained a l l along the column. Recovery of enzyme a c t i v i t y was about 1-2$. But i f the pool was f i r s t incubated with 2 M KCI before the rechromatography, a peak of "low molecular weight" a p u r i n i c endonuclease a c t i v i t y would again be detected ( F i g . 20). The r e s u l t could be explained i f the "low molecular weight species of a p u r i n i c endonuclease had a tendency t o aggregate. A l t e r n a t i v e l y , a p u r i n i c endonuclease could adsorb t o the Sephadex G-100 column and only e l u t e d from the column In the pre-sence of higher KCI c o n c e n t r a t i o n s . When a DEAE pool of Hela c e l l e x t r a c t was analysed by sucrose g r a d i e n t c e n t r i f u g a t i o n , most of the a p u r i n i c endonuclease a c t i v i t y sedimented in the bottom of the c e n t r i f u g e tube; with an S value much bigger than BSA (4.25 S) ( F i g . 21). This agreed with the r e s u l t of the Sephadex G-100 column chromatography. However in an experiment A B C D E F R A C T I O N N O . Figure 18. Sephadex G-100 chromatography of a Hela DEAE pool. The DEAE pool was made t o 2 M NaCl and 35% g l y c e r o l . I t was then incubated a t 0°C f o r 1 hour before put onto the column. The column was e l u t e d w i t h Sephadex b u f f e r X. The markers A-E were dextran b l u e , BSA, 8-IactoglobuI cytochrome C and bromocresol p u r p l e , r e s p e c t i v e l y . 47. 03f-f r a c t i o n n o . Figure 19.. Sephadex G-100. chromatography, of flow-through a p u r i n i c endonuclease a c t i v i t y p u r i f i e d from Peggy c e l l s . The column was e l u t e d with b u f f e r X. The enzyme pool was a p p l i e d onto the column without s a l t - t r e a t m e n t . A A apuri n i c DNA A A n a t i v e DNA 48. F R A C T I O N N O . Figure 20. Sephadex G-100 chromatography of "low molecular weight" form of Hela a p u r i n i c endonuclease. A peak of "low molecular weight" a p u r i n i c endo-nuclease a c t i v i t y was obtained from Sephadex G-100 chromatography of a Hela DEAE pool as shown i n Figure 18. The f r a c t i o n s comprising t h i s peak were pooled and subjected t o Sephadex G-100 chromatography again e i t h e r d i r e c t l y (•* •) o r a f t e r incubation wit h 2.M KCI (• •) f o r an.hour a t 0°C. 49 . - l a c t o g l o b u i i n 16 01 •p C1 .2J >' h >i.oh I-U < 0 .8 l 0.6k 0.4 • 0.21 J I I I L B 1 2 1 B 2 0 2 4 F R A C T I O N N C . Figure 21. Sucrose g r a d i e n t c e n t r i f u g a t i o n of a Hela DEAE pool. The DEAE pool was not s a I t — t r e a t e d . The sucrose gradient. • contained 50 mM Tri s - H C l . (pH.7.5)/0.1 mM DTT. 50. where the DEAE pool was s a l t - t r e a t e d with 2 M KCI p r i o r t o c e n t r i -. f u g a t i o n , - a p u r i n i c endonuclease„activity sedimented in region corresponding t o a molecular weight of 45,000-35,000 ( F i g . 22). No i low molecular weight species was detected. In t h i s experiment, in order t o prevent any reaggregration, the sucrose g r a d i e n t was made 1 M KCI. The peak a c t i v i t y ( f r a c t i o n s number 10-13) from t h i s g r a d i e n t run was pooled and chromatographed on the Sephadex G-100 column a f t e r a 2 M KCI s a l t - t r e a t m e n t . Half of the recovered a p u r i n i c endonuclease a c t i v i t y was again e l u t e d in the low molecular weight f r a c t i o n s ( F i g . 23). I t was t h e r e f o r e concluded the "low molecular weight" species of a p u r i n i c endonuclease was an a r t i f a c t due t o adsorption of the en-zyme t o Sephadex. We found t h a t t h i s adsorption could be e l i m i n a t e d by an e l u t i o n b u f f e r with a high i o n i c s trenght. Thus, e l u t i o n bu-f f e r Y c o n t a i n i n g 1 M KCI was used in subsequent experiments. Ljung-q u i s t and Lindahl (72) had a l s o used an e l u t i o n b u f f e r with a high i o n i c strength (1 M NaCI) t o determine the molecular weight of a p u r i n i c endonuclease from jE. c o I i by Sephadex G-75 column chromatography. A c a l i b r a t i o n of the Sephadex G-100 column e l u t e d with b u f f e r Y was shown in Figure 24. To determine the molecular weight of a p u r i n i c endonuclease a c t -i v i t y f r eed from the complex, Peaks l - l I I were s a l t - t r e a t e d with 2 M KCI. They were then put onto a Sephadex G-100 column which was e l u t e d with b u f f e r Y. B - l a c t o g l o b u l i n (2.5 mg) was a l s o added t o each of the three enzyme pools and served as an i n t e r n a l marker. I t did not have any s i g n i f i c a n t e f f e c t on the endonuclease assay. The r e s u l t of a run of Peak 111 was shown in - F i g u r e 25. The major peak of a p u r i n i c endo-51... 1 2 1 B F R A C T I O N N O . 2 * Figure 22. Sucrose g r a d i e n t c e n t r i f u g a t i o n of a Hela DEAE pool which had been s a l t - t r e a t e d with 2 M KCI before c e n t r i -f u g a t i o n . The sucrose g r a d i e n t contained. 50 mM T-ris-HGI (pH'7.5) /I.M KCI/0.1 mM DTT. Markers A, B and C were BSA, 6-IactogIobuIin and myoglobin, r e s p e c t i v e l y . 52. 2.Cr 15 2 5 3 5 45 F R A C T I O N N O . 23. Sephadex G-100 chromatography of the peak f r a c t i o n s of a p u r i n i c endonuclease a c t i v i t y obtained in the ex-periment described in Figure 22. The peak f r a c t i o n s (number 110-20) of the sucrose gr a d i e n t a n a l y s i s depicted in Figure 22 were pooled and chromatographed on the Sephadex G-100 column a f t e r incubation in 2 M KCI f o r an hour at 0°C. The column was e l u t e d with b u f f e r X. 53. Figure 24. C a l i b r a t i o n of Sephadex G-100 column. The column was e l u t e d with b u f f e r Y c o n t a i n i n g .50 mM Tris-HCl (pH 7.5)/1 M KCI/0.1 mM DTT/10$ g l y c e r o l . Markers A-E were BSA, ovalbumin, g - I a c t o g l o b u I i n , myoglobin and cytochrome C, r e s p e c t i v e l y . 54. p-lactoglobulin 25 f r a c t i o n no. 35 F igure 25. Sephadex G-100 chromatography of s a l t - t r e a t e d Peak III w i th e l u t i o n b u f f e r Y. Peak III was incubated f o r 1 hour w i th 2 M KCI on i c e p r i o r t o chromatography. The column was e l u t e d w i th b u f f e r Y. g -Ia c t o g l o b uI i n was chromatographed toge the r w i th the enzyme pool and served as an i n t e r n a l s i z e marker. 55. nuclease a c t i v i t y was found t o e l u t e s l i g h t l y before 8 - l a c t o g l o - •• b u l i n . A molecular weight of 35,000-40,000 was estimated f o r t h i s ;j species assuming the enzyme was a g l o b u l a r p r o t e i n . Two other minor peaks of a c t i v i t y were a l s o detected. One e l u t e d at f r a c t i o n s c o r -responding t o a molecular weight of 70,000-75,000, p o s s i b l y a dimer of a p u r i n i c endonuclease or an enzyme complex of o t h e r e n t i t y . An-other a c t i v i t y e l u t e d in the v i c i n i t y of cytochrome C. I n t e r e s t i n g l y , UV endonucleases of M. luteus were reported t o have a molecular weight of 10,000-15,000 (67). For Peak I I , most of the a p u r i n i c endonuclease a c t i v i t y recover-ed e l u t e d a t t r a c t i o n s corresponding t o a molecular weightvof 22,-000-25,000 ( F i g . 26). In the case of Peak I, the presence of KCI In the column f r a c t i o n s created a problem. Because of the low level of enzyme a c t i v i t y in t h i s p o o l , 5-10 uI of a l i q u o t s of each column f r a c t i o n was needed f o r the endonuclease assay. 5 pi of a column f r a c t i o n would introduce 100 mM KCI i n t o the assay mixture with a f i n a l volume of 50 u l . As discussed e a r l i e r , high concentrations of KCI were i n h i b i t o r y - t o the a p u r i n i c endonuclease a c t i v i t y in Peak I. Thus, the column f r a c t i o n s were f i r s t d i a l y s e d in 1 I. of 50 mM T r i s - H C l (pH 7.5)/0.1 mM DTT/ 10 % g l y c e r o l f o r 2-3 hours before they were used f o r enzyme assays. With t h i s m o d i f i c a t i o n , a p u r i n i c .endonuclease a c t i v i t y from Peak I was found t o e l u t e as a broad peak ( F i g . 27). I t was i n f e r r e d a p u r i n i c endonuclease with a molecular weight of 45,000-50,000 was e l u t e d in the f i r s t h a l f of the broad peak. The other h a l f of the broad peak was composed of enzyme species w i t h a molecular weight s i m i l a r t o 56. Figure 26. Sephadex G-100 chromatography of s a l t - t r e a t e d Peak II with e l u t i o n b u f f e r Y.-Peak I I was prepared and chromatographed as Peak III in Figure 25. 57. Figure 27. Sephadex G-100 chromatography of s a l t - t r e a t e d Peak I with e l u t i o n b u f f e r Y. Peak I was prepared and chromatographed as Peak III in Figure 25. 58. those of Peak I I . The experiment provided f u r t h e r evidence f o r a p o s s i b l e conversion of Peak I t o Peak II and Peak III upon phophos-c e l l u l o s e rechromatography. 59. DISCUSSION 1. Comparison of a p u r i n i c endonuclease a c t i v i t y in Hela c e l l s and human f i b r o b l a s t s : (a) General p r o p e r t i e s : In agreement with another report (42), we found t h a t a c t i v i t y of a p u r i n i c endonuclease in crude e x t r a c t s (high-speed supernatant) of Hela c e l l s was s i m i l a r t o t h a t of nor-mal human f i b r o b l a s t s . The s p e c i f i c a c t i v i t y of a p u r i n i c endonu-cle a s e in crude e x t r a c t s of Hela c e l l s was in the range of 400-800 units/ug of p r o t e i n . The value reported f o r normal human f i -b r o b l a s t s was between 380-670 units/ug p r o t e i n s (44). The a p u r i n i c endonuclease a c t i v i t y in Hela c e l l s was then r e -solved i n t o three peaks of a c t i v i t y by phosphoceI IuIose column chro-matography. They were designated as Peaks I, I I , I I I . They had a 2+ s i m i l a r pH optimum and Mg requirement as the enzyme species of hu-man f i b r o b l a s t s . The enzyme species of human f i b r o b l a s t s were f u r t h e r reported to be stimulated t o 2.5-fold by 10 mM KCI. They had a ha I f - l i f e of 6 min at 45°C in 230 mM KP0 4 (pH 7.4). But in Hela c e l l s , a l l three enzyme species were i n h i b i t e d by i n c r e a s i n g concentrations of KCI and NaCl, except Peak i l l which was only s l i g h t l y s t i m u l a t e d by 20-40 mM KCI ( f i g . 1 5 ) . Peaks l - l I I of Hela c e l l s were d i f f e r e n t in t h e i r t h e r m o s e n s i t i v i t i e s . Peak III was most h e a t - l a b i l e , i t s h a l f -l i f e ( t ' 1 / 2 ) a t 45°C i n 50 mM KP0 4 (pH 7.4) was only 2 t o 3 min. L i t t l e loss of enzyme a c t i v i t y was observed f o r Peak I under these c o n d i t i o n s . P r e l i m i n a r y experiments i n d i c a t e d t h a t both species were 60. more h e a t - s e n s i t i v e in 250 mM KP0 4 (pH 7.'4); at 45°C, t h e i r ha I f -l i v e s were less than 1.5 min. In E_. co I i , a minor species of apu-r i n i c endonuclease, endonuclease IV, was found t o be s t a b l e a t 45°C (27). Endonuclease IV however had no Mg 2 + requirement and was f u l l y a c t i v e in the presence of EDTA. Peak II of Hela c e l l s seemed to c o n s i s t of a h e a t - l a b i l e ( + i / 2 = ^ m ' n ) a n ^ a m o r e h e a t - s t a b l e ( t ^ 2 = 25 min) components. Thus, a p u r i n i c endonuclease a c t i v i t y of human f i b r o b l a s t s and Hela c e l l s d i f f e r in t h e i r t h e r m o s e n s i t i v i t i e s and i n h i b i t i o n by i n -cr e a s i n g s a l t c o n c e n t r a t i o n s . Whether these d i f f e r e n c e s in p r o p e r t i e r e f l e c t the n e o p l a s t i c nature of Hela c e l l s remains a q u e s t i o n . I t i s however not uncommon t h a t isoenzymes p u r i f i e d from d i f f e r e n t t i s -sues o r organs have d i f f e r e n t p r o p e r t i e s . For example, while the ap-u r i n i c endonuclease in c a l f - l i v e r has a pH optimum of 9.5 and an op-2t timal Mg concentration of 0.01-0.05 mM, the corresponding values f o r the caIf-thymus enzymes are 8.5 and 0.5-3 mM r e s p e c t i v e l y . Fur-thermore, the a c t i v i t y of the calf-thymus enzyme i s s t i m u l a t e d by 0.04 M NaCI and t h a t of c a l f - l i v e r i s i n h i b i t e d t o 50% by 0.025 M NaCI (29). This i s a l s o evident when a p u r i n i c endonuclease i s o -lated from human f i b r o b l a s t s and placenta are compared. For exam-p l e , the p l a c e n t a l enzymes are o p t i m a l l y a c t i v e at around 3 mM MgCI 2 and those of human f i b r o b l a s t s have an optimum of 10 mM MgC^-(b) R e l a t i v e p r o p o r t i o n of flow-through and h i g h - s a l t e l u a t e species  of a p u r i n i c endonuclease a c t i v i t y : In normal human f i b r o b l a s t s , a c t i v i t y of the flow-through species was about 20-30$ t h a t of h i g h - s a l t e l u a t e species. In Hela c e l l s , a c t i v i t y of Peak I ( f l o w -6 1 . through a c t i v i t y of Hela c e l l s ) was only 2-4$ the a c t i v i t y of Peak • III ( h i g h - s a l t e l u a t e a c t i v i t y of He I a ceI I s ) . To see i f t h i s re-presented a p e c u l i a r c o n d i t i o n of Hela c e l l s , we had p u r i f i e d ap- -u r i n i c ednonuclease a c t i v i t y from a supposedly normal c e l l l i n e of human f i b r o b l a s t (Peggy c e l l ) . The r e l a t i v e p r o p o r t i o n of flow-through and h i g h - s a l t e l u a t e a c t i v i t i e s was s i m i l a r t o t h a t in Hela eel Is. We do not know the reason f o r t h i s discrepancy. Perhaps the flow-through a c t i v i t y i s subjected t o c e l l u l a r metabolic r e g u l a t i o n which may be a f f e c t e d by t i s s u e c u l t u r e c o n d i t i o n s . The c e l l u l a r l e v e l of another DNA r e p a i r enzyme, p h o t o r e a c t i v a t i n g enzyme, was claimed t o be a f f e c t e d by composition of the t i s s u e c u l t u r e medium (73). I t was found t h a t human f i b r o b l a s t s grown in Eagle's minimal, es-s e n t i a l medium contained very low l e v e l s of p h o t o r e a c t i v a t i n g enzyme compared t o eel Is grown in Dulbecco's modified Eagle's minimal me-dium. We had r o u t i n e l y supplemented our t i s s u e c u l t u r e medium with several a n t i b i o t i c s while such was not a p r a c t i c e in the e a r l i e r s t u d i e s with human f i b r o b l a s t s (44,45). 2. Interconversion of flow-through and h i g h - s a l t e l u a t e species of  a p u r i n i c endonuclease from Hela c e l l s : The r e s u l t of the phosphoceI IuIose rechromatography experiment suggested t h a t the flow-through and the h i g h - s a l t e l u a t e species of a p u r i n i c endonuclease in Hela c e l l s are i n t e r c o n v e r t i b l e . I t w i l l be of i n t e r e s t t o i d e n t i f y the f a c t o r s governing such i n t e r c o n v e r s i o n . The study may u l t i m a t e l y lead t o an understanding of the r e p a i r de-f e c t in XP-D c e l l s , s i n c e they are d e f i c i e n t in the flow-through 62. s p e c i e s o f a p u r i n i c e n d o n u c l e a s e . One can p o s t u l a t e t h a t a d s o r p t i o n of h i g h - s a l t e l u a t e a c t i v i t y t o t h e phosphoceI I u I o s e column i s i n -h i b i t e d by ; a f a c t o r E. F l o w - t h r o u g h a c t i v i t y t h e r e f o r e i s a com-p l e x of E and t h e h i g h - s a l t e l u a t e s p e c i e s o f a p u r i n i c e n d o n u c l e a s e . T h i s complex w i l l d i s s o c i a t e upon p h o s p h o c e l I u l o s e chromatography. An a n a l o g y i s t h e sigma f a c t o r (a) i n E_. coj_i_ w h i c h u s u a l l y forms a complex w i t h RNA po l y m e r a s e . I t i s r e q u i r e d f o r t h e i n i t i a t i o n o f RNA s y n t h e s i s . The sigma f a c t o r can be s e p a r a t e d from t h e enzyme by chromatography on phosphoceI I u l o s e (74,75). Once f a c t o r E i s i s o l a t e d from human f i b r o b l a s t s , we s h a l l t e s t w h e t h e r a d d i t i o n o f t h i s f a c t o r t o t h e h i g h - s a l t e l u a t e a p u r i n i c en-d o n u c l e a s e a c t i v i t y from XP-D c e l l s w i l l r e s u l t i n t h e f o r m a t i o n o f any f l o w - t h r o u g h a c t i v i t y . T h i s k i n d o f e x p e r i m e n t w i l l d e t e r m i n e whether i n XP-D c e l l s t h e r e i s a d e f e c t i n f a c t o r E o r i n i t s p r o d -d u c t i o n , o r whether t h e r e Is a d e f e c t i n a p u r i n i c e n d o n u c l e a s e w h i c h p r e v e n t s t h e a s s o c i a t i o n o f t h e enzyme w i t h f a c t o r E. 3. M o l e c u l a r w e i q h t d e t e r m i n a t i o n s o f Peaks l - l I I .: When Peaks l - M I were a n a l y s e d by Sephadex G-100 column c h r o -matography, a major p a r t o f each o f t h e t h r e e enzyme a c t i v i t i e s was foun d t o be a s s o c i a t e d w i t h a h i g h m o l e c u l a r w e i g h t c o m p l e x . T h i s r e s u l t was c o n f i r m e d i n d e p e n d e n t l y by t h e s u c r o s e g r a d i e n t sediment-a t i o n a n a l y s i s . P r e l i m i n a r y e x p e r i m e n t s w i t h Sephadex G-200 column chromatography i n d i c a t e d t h a t t h e complexes a r e l e s s t h a n T50,000 i n l e c u l a r w e i g h t . T h i s a g a i n does n o t seem t o be a p e c u l i a r p r o p e r t y o f H e l a c e l l s . We o b t a i n e d s i m i l a r r e s u l t from f i b r o b l a s t s o f a 63. normal human c e l l l i n e , Peggy c e l l s . A l s o , during the e a r l i e r stages of enzyme p u r i f i c a t i o n from the human lymphoblastic c e l l l i n e CCRF-CEM (76) and E. c o l i (26), a p u r i n i c endonuclease a c t i v i t y was reported t o be associated with high molecular weight complexes. However, with the same p u r i f i c a t i o n procedures as ours, Kuhn l e i n et aj_. (45) reported t h a t flow-through a p u r i n i c endonuclease a c t i v i t y had a S value of 3.3, s l i g h t l y l a r g e r than the h i g h - s a l t e l u a t e species which had a S value of 2.8. The two S values cor-respond t o a molecular weight of around 40,000 and 35,000 r e s p e c t i -v e l y , i f one assumes, t h a t a p u r i n i c endonuclease i s a g l o b u l a r p r o t e i n . No high molecular weight complex was detected. In these experiments, the two enzyme species were st o r e d f o r a period of more than 1-2 months before a n a l y s i s (U. Kuhnlein, personal communication). Pre-sumably, over t h i s length of time, the a p u r i n i c endonucleases had d i s s o c i a t e d from the high molecular weight complexes. Subsequently, we found t h a t a p u r i n i c endonuclease a c t i v i t y could be d i s s o c i a t e d from the high molecular weight complex by making the en-zyme s o l u t i o n 2 M KCI o r 2 M NaCl. The major a p u r i n i c endonuclease i n Peak III had a molecular weight of 35,000-40,000. Those of Peak II were s m a l l e r with a molecular weight of 22,000-25,000. Peak I seemed t o contain 2 kinds of a p u r i n i c endonuclease, one with a molecular weight of 45,000-50,000 and the other with a molecular weight s i m i l a r t o those of Peak I I . Limited by the r e s o l u t i o n of the Sephadex G-100 column, we could not conclude whether the enzyme species in Peak I were l a r g e r than the corresponding h i g h - s a l t e l u a t e species. Another question which remained unanswered i s whether the assoc-64. i a t i o n of a p u r i n i c endonuclease with a high molecular weight complex • has any b i o l o g i c a l s i g n i f i c a n c e o r i s merely a d v e n t i t i o u s . In t h i s re-gard, i t i s of i n t e r e s t t o note t h a t a p u r i n i c endonuclease p u r i f i e d from the p l a n t embryo Phased us m u l t i f l o r u s i s a nonhistone p r o t e i n of chromatin (31). The a s s o c i a t i o n of a p u r i n i c endonuclease with other accessory p r o t e i n s may be important f o r i t s in v i v o f u n c t i o n . The high molecular weight complex may be par t of a r e p a i r machinery o r represent a storage form of a p u r i n i c endonuclease in the cytoplasm. 4. Conclusion : Three species of a p u r i n i c endonuclease a c t i v i t y were found in Hela c e l l s , i n c l u d i n g a flow-through s p e c i e s . For some yet uniden-t i f i e d reason, we got very l i t t l e flow-through a p u r i n i c endonuclease a c t i v i t y from e i t h e r Hela c e l l s o r normal human f i b r o b l a s t s . Ap-u r i n i c endonuclease a c t i v i t i e s from Hela c e l l s d i f f e r e d in some r e s p e c t s , such as t h e r m o s e n s i t i v i t i e s , from those of human f i b r o -b l a s t s . Aside from these d i f f e r e n c e s , we t h i n k Hela c e l l s w i l l provide enough enzyme material f o r f u r t h e r s t u d i e s of the f o l l o w i n g problems: (1) the i n t e r - r e l a t i o n s h i p s between the d i f f e r e n t species of a p u r i n i c endonucleases, (2) the b i o l o g i c a l s i g n i f i c a n c e f o r the a s s o c i a t i o n of a p u r i n i c endonuclease with a high molecular weight compI ex. 65. BIBLIOGRAPHY 1. L i n d a h l , T. and Nyberg, B. (1972) Rate of depurination of n a t i v e deoxyribonucleic a c i d . Biochem. 3610-3617. 2. 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