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Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells
154
Biochimica et Biophyslca Acta, 442 (1976) 154--161 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98652 REPAIR OF X-RAY-INDUCED SINGLE STRAND BREAKS IN TOLUENIZED ESCHERICHIA COLI CELLS
EVELYN A. WALDSTE]N a~ and R.B. SETLOW b a Tel Aviv University, Genetics Unit, Department of Botany, The Dr. George S. W/se Center for Life Seiencs, Tel Aviv (Israel) and b Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.) (Received January 28th, 1976)
Summary
W e have used sedimentation in alkalito estimate the repairof X-ray-induced single strand breaks in the D N A of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except A T P although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide ( N M N ) inhibits repair in wild type cells we interpretthe resultsas indicatingthat both ligase and polymerase I are needed for repair but that the amount of any gap fillingis small and extensive repair replicationis not necessary.
Introduction Ionizing radiation makes large numbers of single strand breaks in D N A . In most cells the breaks are repaired rapidly and the average break contributes littleto lethality.However, bacterial cellsdefective in polymerase I have a decreased ability to repair s~igle strand breaks and are more radiation sensitive than wild type cells(for review see ref. 1). In vitro experiments show that repair of strand breaks requires more than just a functioning system of D N A polymerase I plus ligase [9.--4].But D N A polymerase I and ligase plus an assembly of cofactors can rejoin up to 50% of single strand breaks and partiallyrestore the transforming activity of Bacillus subtil~s D N A in vitro of D N A that had been irradiated in vivo [5]. Enzymes plus cofactors are necessary to repair some of the chain breaks resulting f ~ m
* TO whom ¢on~ondence should he sent,
the decay of 3~p [6]. In cells reudered permeable by toluene txeatment the addition of exogenous enzymes is not necessary for Rpair but numy cof~tors are [7,8]. A variety of cofactors are needed to repair c h ~ ~ in nuclei of mammalian cells [9]. In cells rendered permeable by toluene treatment, ATP is ne~.ed for the
non-conservative Rmthasis observedin polA cells following ultraviolet ~adia. tion [10--12]. Such synthesis is difficult to detect in wild type cells because of the high levels of semi~onservative synthesis that is stimulated by ATP. Moreover, following ultraviolet irradiation, ATP stimulates the a ~ ef strand breaks that are mediated by the uvrA + uvrB gene produc~s [13]. On the other hand, ATP is not required for the repair type synthesis ~a~ follows treatment of toluenized cells with DNAase I [14]. We were interested in r n e ~ repa~ of ionizing radiation damage in toluenized cells. However, the amount of repa~ replication after irradiation of wild type Escher~ch~a coli is alm~t ~ml~e~ly masked by the production of new origins of replication [15]. Hen~, we h a ~ confined ourselves to measuring the repair of sin~1o strand breaks in toluen~ed cells and the effects of cofactors on such repair. We observed that strand break repair does not require all the cofactors n~essary for the demonstration of repair replication. Methods The cells used in this work were E. coli KMBL 1056 F -thyA 301 endA 101 (wild), KMBL 1787 F- thyA 301 argA 103 bio-87 pheA 97 endA 101 polAI and KMBL 1795 F" thyA 301 argA 103 bio-87 pheA 97 mete 72 strA endA 101 resA 1 obtained from R. Ben.Ishai. Cultures were grown in M9 medium supplemented with 0.1% glucose, casamino acid (2.5 mg/ml), thymidine (5/~g/ml) and thhmine (0.2 ~gtml). Amino acids and biotin, when required, were added to 50 and 2 ~glml, respectively. To label the bacterial DNA[SH]thymidine (2.5 ~Ci[ml of i l . 5 Ci/mM, Nuclear Research Center, Beer Sheva) was added to the medium for three doubling times. Exponentially growing cells at density of 0.5-109--1 • 109 per ml were collected by centrifugation, resuspended at 5 • 109 c~Jis per m| in 50 mM potassium phosphate buffer, pH 7.4, and treQt~d with ~luene as described by Moses and Richardson [14]. The toluen~ed ceUs were cooled to 0°C, washed by sedimentation with four volumes of potas~um ~o~phate buffer, resuspended and irradiated with app~x. 5000 tad of X-rays from a Phillips MG ~ 00 X-ray machine. Following irradiation, the cells were incubated at 30~C for th-nes up to 20 rain in solutions containing 70 mM potassium phosphate buffer and 7 v~M Mg2.. Other addition~ when used, are indicated in the text. Tl~re was no s e ~ g of X-ray induced breaks nor any breakdown in cells kept at 0 °C for 20 rain. At the end of the incubation period cells were traudormed into s p h e ~ l~ and DNA sedimented in alkaline sucrose gradients as described by Rupp Howa~-Fianders [16]. Sedimentation was c a n e d out in an SW 50.1 rotor of a Beckman L5-50 centrifuge spun at 30 000 rev.~in for 90 rain at ~ P C Fractions were collected from the top of the gred~nt and the d~st~bu~n of radio. activity throughout the gradient was determined as described ~ w h e ~ [17].
156 Resul~
The -:ist~nce sedimented by DNA in alkali depends on the single strand molecular weight. We interpret increases (decreases) in sedimentation cor~tant as decreases tincre~u~s) in the numbers of single strand breaks. Our basic observation is shown in Fig. 1. The single strand breaks appearing in the D N A of irradiatedwild type cellsare only slightlyrepai~d if the cellsm ~ incubated in the absence of ATP, but are almost completely repaired if the cells are incubated in the presenc~ of A T P (no other cofactors present). The rate of repair is rap.~das is indicated in Fig. 2 and the repair observed in the presence of A T P is inhibited by N M N , an inhibitor of polynucleotide lig~se. These data suggest that the rejoininginvolves ligaseactivityand the data in Fig. 3, showing that there isno rejoiningin polA cellsincubated in the presence of A T P indicate that polymerese I is also necessary for rejoining.The stimulation of rejoining by A T P is not an artifactof an irreversiblebreakdown of D N A in its absence, because A T P may be added up to 5 rain after the start of incubation at 30°C and stillgive just about complete rejoining (Fig. 4). W e have also made the following observations on irradiated wi~i type cells (data not shown). The optimal A T P concentration for strand rejoining in 9.0 rain is 1.3-d).65 raM. At 0.13 m M rejoining is only half completed. The rejoining reaction needs M g 2÷, N A D , the cofactor of polynucleotide ligasehas littleinfluence on break rejoiningin the presence or absence of ATP. W e wanted to find out whether the enhancing action of A T P on strand break repair was specific or was a general property of other ribonucleotide triphos-
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Pig. 1. Alkaline sucrose gradient sedimentation profiles of DI~A from E. c o l i KMBL, 1056 (wild type). A no irradiation, 20 rain incubation: t , irradiation, no ineubat/on; o. inradiation. 20 m i n i n e u b a t i o n , no additions; e, irradiat/on, 20 rnin incubation PlUS ATP (1.3 mM). The molecular weight scale was derived as in ref. 27.
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Fig. 2. Alkaline sucrose gradient sedimentaUon p~omes o~ DNA f~,cm E. co~ KMBL 1056 (wnd type), A11 samples irradiated, POst-irradiation conditions: o, 20 rain, n o addi~ons: ®, 10 r n ~ p t ~ ATP (1.3 ~h~)~ e. 20 m|n PlUS ATP ~.1.3 raM): ~. 20 rain plus NMN (5 mM) plus ATP (1.3 InM).
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Fig. 3. , c J k ~ n e sucrose gradient sedimentaUon profiles o f DNA from E. coli KMBL 178"/polA. s , n o irradiation, 20 rain incubation, n o additionS; ~, inadiation, n o incubation; o, is~adi~tion, 20 mh~ incuba#./on. n o additionS; @4 in'adiatlon. 20 min i n c u b ~ o n plus ATP (1.3 mM). S i m ~ red,tits have been obtained with DNA frow E. coil KMBL 1795 m A .
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Fig. 4, Alkaline ~etose gradient secUmentatic~n profiles of DNA from E. coil KMElr, 1056 (wild type). Post-in'adlation incubation for 20 min~ o, n o additiolm~ e~ ATP (1.3 raM) added 2.5 rain after start of incubation- ~, ATP (1,3 raM) added 5 rain after start o f incubation.
phates. W e found (Fig. 5) that C T P and G T P are as good as AT~ ~,and that U T P does not stimulate at all (data not shown). Hence, the A T P effect is not specific for that t:,qphosphate,Moreover, the other individualdeoxynucleoside triphosphates also stimulate rejoining in the absence of A T P (Fig, 6a). The best rejoining observed is in the presence of allfour dNTPs plus N A D (Fig. 6b).
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F/g, 5, Alkaline sucrose gradient sedimentation p:~flles of DNA from F, e c l i KMBL 1056 (wild type). Post-irradiation ineub~ttion for 20 rain; o, no additians~ o, CTP (1,3 mM); ^, GTP (0.65 raM).
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Fig. 6,(a and b) Alkaline sucrose gradient sedimentation profiles of DNA ft~m E. coli KMBL 1056 (wikl type). Post*Irradiatlon incubation f o l 20 min; o, n o additions: o, dATP (1~0 ~M): o, TTP (130 ~M)~ ~, four dNTPs (33/JM each) plus NAD (3.3 mM): A, four dDNTPs plus NAD plus ATP (1.4 raM),
Discussion Our data indicate that in toluenized wild type cellsthe re~inh~g of strand breaks resulting from X-irradiation is stimulated by A T P or by other NTPs (except U) or dNTPs. Since the stimulatingeffect of A T P isinhibited by N M N (nicotinarnide mononucleotide), polynucleotide ligase is implicated in the joining process as is to be expected. W v interpret the fact that N A D , the cofactor for ligase,stimulatesthe rejoining process only slightlyin the presence or absence of ATP, as indicatingthat there m a y be sufficientendogenous N A D to accomplish rejoining. L o w levelsof endogenous cofactors m a y be important in the rejoining process because the n u m l ~ of breaks to be joined is relatively small, perhaps of the order of 100 per chromosome. The effect of N A D in the E. colt system are different from those observed in toluenized B. subtU~ in the latterN A D is a strong stimulator of rejoining [18]. Polynucleotide ligase is not sufficient by itselfto rejoin breaks since the addition of N A D in the absence of A T F does not lead to rejoining. Another necessary enzyme system seems to be pol.w~erase I since we observe no rejoining in poIA mutants when A T P is added. This inabilityof poIA cellsto rejoin s~ugle strand breaks does not result from the failure of A T P to enter the toluen~ed cells because in polA cells,as in wild tylm.• cells [13], A T P stimulates ~ e production of single strand breaks following ultravioletirradiation. Not m u c h data exist on conditions of strand break rejoining'~nsemi-permeable cells. Repair synthesis, nct accompanied by break rejoining, has bee~ observed in X-i~adiated polA mutants of E. co/i (19). X-zay treatment induces some reduced non-conservative D N A sFnthesis in polymerase I.defieient mutants of B. subtil~s [20] whereas no ~toration of tmmforming ~ was
observed in similar experiments [8]. Extensive ("long patch") polymerase H and III dependent repair replication was found in ultraviolet-~radiated poty-
160 merase I mutants of E. coli [10--12]. But the poIA independent repair synthesis is not followed by a ligation reaction if incision breaks are allowed to accumulate [21]. Thus, D N A polymemse I seems to be the 0nly polymerase able to take part in break rejoining in semi-permeable cellsalthough two types of break repair mechanisms were shown to existin vivo [22]. Toluenized cells possibly loose some factor necessary for break repairby the second mechanism. The stimulatingeffect of A T P or other nucleoside triphosphates for rejoining of breaks is not easy to explain. In X-irradiated cellsA T P does not seem to stimulate an incision step. Even in poIA cellswhere strand rejoining is lacking no more breaks are found in the presence of A T P than in its absence. Thus the stimulating effect of A T P or other triphosphates m a y have to do with the gap filling process it.ll or a process closely related to it, especially since the numbers of gaps to be filledis small and small pools of endogenous factors m a y almost be sufficientto accomplish rejoining. A T P partiallystimulatesthe nonconservative repair type synthesis occurring in 7-irrediatedB. subtilis cells [20] but has no effect on the restoration of the transforming activityand molecular weight of its D N A [7,8]. Studies that measure repair synthesis are not directly comparable with ours since A T P and allfour dNTPs are used in them, but only A T P is needed for extensive strand rejoiningin toluenized wild type cells. The effect of A T P or other ~iphosphates on strand rejoiningafter X-irradia. tion is very different from its effects on ultraviolet-irradiatedpermeabilized cells. In the latter it stimulates the appearance of single strand breaks by a process that seems to be mediated by the uvrA + uvrB gene products [13]. Moreover, A T P is needed for repairsynthesis in ultraviolet.irradiat~_~d poIA cells [10--12]. The stimulatory effects of the various triphosphates for strand rejoining show more analogy to the repair following nuclease treatment of toluenized cells [14]. In this system no repair type synthesis was observed in poIA cells, and A T P was not necessary for repair synthesis in wild type ceils, but, of course, the ot~herdl'PrPs were present to measure such synthesis. The amount of repair replication following ionizing nldiation has not been estimated directly in E. coli. Its amount depends on the endogenous level of ligese [23]. High amounts of ligaselimit the amount of nucleotide insertion.In mammalian cellsthe numbers of nucleotides added per chain break produced is about of 2--3 [24--271. Despite our lack of knowledge about the detailsin the processes observed, our data indicate that there,is no need for extensive repair replication in the strand rejoiningin toluenized cells.Our observations are consistent with the idea that following X-irradiationthe ends of the broken regions must be "tailoredand one or a few nucleotides mlded by polymerase I, followed by ligation. Acknowledgments This investigation was sponsored by the Jewish Agency through the Israeli Center of Absorption in Science. W e are indebted to R. Ben-lshai for supplying bacterial strains and S. Manoilis for perforrning some of the experiments. This work was carried out, in part, at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Develol~ment Administration.
References 1 Set~ow, R,B. and Setlow, J.K. (1972) Ann. Rev. B~ophys. ~ , 1, 2~3--34~; 2 Kapp, D.S. an8 Smith, K.C. (1969) |nt. J. RaciSt, ~Ioi, 14, ~ 7 ~ T | 3 JaeobQ, A,, Bopp, A. ~ d H~[en, U. (t972) In|. J. ~a~.iat. ~ . 22~ 431--43~ 4 I~ndbeck, L. 8,nd H a p n , U, (1973) B l o c h ~ . ~ b y s , A ~ 331, 3 ~ - 4 2 7 ~) L~lpis~ P,J. and Gim¢~,'~, A,F, (1972) Pro~. NaU. A©ad. Sci. U,S. 69, 3=11--3214 fJ Fodo~, I,, Matvlenko, N,|,, Zlot~d~ov. K,M,, T a Q y ~ , V,L, S ~ n , A.S. ~ ~ v A~ {1)7~ Nucleic Acid R ~ , 2, 63r)~646 7 Nogutl, T, and Kada, T. (1972) J. Mo]. Biol. ~?, I"~07--512 8 N o g t l , T. and Kada, T, (197~)) ~OChi~l. Biophy~. Ae([& 3~5, 2 ~ - - ~ 3 9 Matmldalta, H, and I ~ r u n o , I, (I~)72) Biochim, ~ p h y s . A ¢ ~ ~'~2. 2 0 ~ - - 2 | I 19 Masker, W.E, an(1Ha~w~It, P.C, (19~3) Pro~, Na~. A©~d, SoL U,S, ~O, 1 ~ 1 3 3 11 M ~ k c r , W.E,~ H~naw~dt, P,C, and Shizuy~. H, (19#3) Nal, New ~ c ] . ~ 4 , ~ 2 ~ 3 12 Masker, W.E, an0. H a n a w ~ t , P C , (~974) ~. MoL ~ o l , 8 8 , 1 3 - - 2 3 13 Waldstein, E.A., Sharon, 11. and Bert.l~h~, R, (1974) P~o~. N ~ , A e ~ , S~i, ~,S, #I~ ~ b 1 ~ 14 Moses, R,E, ~ I d R i c h a ~ i ~ n , C.C, (19"~0) PI,~, Natl, Ac~l, SoL ~LS. 6~. 6 " ~ 4 - ~ I 15 Billen, D, (1969) ,~, Baeteriol, 9#, 1169.-1175 16 Rupp, W,D, ~nd ~ow&~rd-Fl~ders, P, ( I ~ 8 ) J. M ~ , ~ , 31, ~ I ~ 0 ~ 17 Carrier, W,L and Setlow, R,B. (1971) A~a]. ~ ¢ h ~ , 43, 4 2 # - ~ 3 2 18 B~len, D, and H e l I e r m ~ G,R, (19~'4) |~ochlm. ~ o p h y s . A c ~ 383, 3 7 ~ - - 3 ~ 19 Gazicv, A,J., Sert~eva, S,A, and Kuzin, A,M. (1974) l ~ c . A c ~ . S~L ~LS.S~R., ~ , S~L ~, 49~--497 2 0 Billen~ D. a~.',. Hellerm~nn, G.R, (1974) 3 1 o c h ~ . ~ s . A~a 3~, I~5--I~ 21 Sharon~ R.0 Miller, C, ~md Ben-lsh~, R, (1975) J. B ~ r ~ o l , 123,11~'--111~ 22 Town, C,D., Smith, K,C, and KaI~lan, H,S, (1971) Sc~r~ce 1~2, ~ 5 1 - - ~ 23 B ~ I ~ , D., H e l l e ~ a n n , G,R, ~ d StalUo~, D,R, (19~5) J, B ~ e ~ , I~4, ~ 24 Painler~ R,B, ~ d Yown~, B.~, (1972) M:z~aL Res. 14, 225--235 2 5 Fox, M, and Fox, B,W, (1973) Int, J, Radi~t. Bi~d. 23,333.--~5~ 26 Donl~n, T. and Norman, A, (1971) Mut~t, Re~ 13, 9"~--I07 27 Rel[~n~ 3,D, an0 SeUow, B,~I, ~1974) C ~ c e r Re~. 34, 3318--3325
Biochimica et Biophyslca Acta, 442 (1976) 154--161 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98652 REPAIR OF X-RAY-INDUCED SINGLE STRAND BREAKS IN TOLUENIZED ESCHERICHIA COLI CELLS
EVELYN A. WALDSTE]N a~ and R.B. SETLOW b a Tel Aviv University, Genetics Unit, Department of Botany, The Dr. George S. W/se Center for Life Seiencs, Tel Aviv (Israel) and b Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.) (Received January 28th, 1976)
Summary
W e have used sedimentation in alkalito estimate the repairof X-ray-induced single strand breaks in the D N A of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except A T P although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide ( N M N ) inhibits repair in wild type cells we interpretthe resultsas indicatingthat both ligase and polymerase I are needed for repair but that the amount of any gap fillingis small and extensive repair replicationis not necessary.
Introduction Ionizing radiation makes large numbers of single strand breaks in D N A . In most cells the breaks are repaired rapidly and the average break contributes littleto lethality.However, bacterial cellsdefective in polymerase I have a decreased ability to repair s~igle strand breaks and are more radiation sensitive than wild type cells(for review see ref. 1). In vitro experiments show that repair of strand breaks requires more than just a functioning system of D N A polymerase I plus ligase [9.--4].But D N A polymerase I and ligase plus an assembly of cofactors can rejoin up to 50% of single strand breaks and partiallyrestore the transforming activity of Bacillus subtil~s D N A in vitro of D N A that had been irradiated in vivo [5]. Enzymes plus cofactors are necessary to repair some of the chain breaks resulting f ~ m
* TO whom ¢on~ondence should he sent,
the decay of 3~p [6]. In cells reudered permeable by toluene txeatment the addition of exogenous enzymes is not necessary for Rpair but numy cof~tors are [7,8]. A variety of cofactors are needed to repair c h ~ ~ in nuclei of mammalian cells [9]. In cells rendered permeable by toluene treatment, ATP is ne~.ed for the
non-conservative Rmthasis observedin polA cells following ultraviolet ~adia. tion [10--12]. Such synthesis is difficult to detect in wild type cells because of the high levels of semi~onservative synthesis that is stimulated by ATP. Moreover, following ultraviolet irradiation, ATP stimulates the a ~ ef strand breaks that are mediated by the uvrA + uvrB gene produc~s [13]. On the other hand, ATP is not required for the repair type synthesis ~a~ follows treatment of toluenized cells with DNAase I [14]. We were interested in r n e ~ repa~ of ionizing radiation damage in toluenized cells. However, the amount of repa~ replication after irradiation of wild type Escher~ch~a coli is alm~t ~ml~e~ly masked by the production of new origins of replication [15]. Hen~, we h a ~ confined ourselves to measuring the repair of sin~1o strand breaks in toluen~ed cells and the effects of cofactors on such repair. We observed that strand break repair does not require all the cofactors n~essary for the demonstration of repair replication. Methods The cells used in this work were E. coli KMBL 1056 F -thyA 301 endA 101 (wild), KMBL 1787 F- thyA 301 argA 103 bio-87 pheA 97 endA 101 polAI and KMBL 1795 F" thyA 301 argA 103 bio-87 pheA 97 mete 72 strA endA 101 resA 1 obtained from R. Ben.Ishai. Cultures were grown in M9 medium supplemented with 0.1% glucose, casamino acid (2.5 mg/ml), thymidine (5/~g/ml) and thhmine (0.2 ~gtml). Amino acids and biotin, when required, were added to 50 and 2 ~glml, respectively. To label the bacterial DNA[SH]thymidine (2.5 ~Ci[ml of i l . 5 Ci/mM, Nuclear Research Center, Beer Sheva) was added to the medium for three doubling times. Exponentially growing cells at density of 0.5-109--1 • 109 per ml were collected by centrifugation, resuspended at 5 • 109 c~Jis per m| in 50 mM potassium phosphate buffer, pH 7.4, and treQt~d with ~luene as described by Moses and Richardson [14]. The toluen~ed ceUs were cooled to 0°C, washed by sedimentation with four volumes of potas~um ~o~phate buffer, resuspended and irradiated with app~x. 5000 tad of X-rays from a Phillips MG ~ 00 X-ray machine. Following irradiation, the cells were incubated at 30~C for th-nes up to 20 rain in solutions containing 70 mM potassium phosphate buffer and 7 v~M Mg2.. Other addition~ when used, are indicated in the text. Tl~re was no s e ~ g of X-ray induced breaks nor any breakdown in cells kept at 0 °C for 20 rain. At the end of the incubation period cells were traudormed into s p h e ~ l~ and DNA sedimented in alkaline sucrose gradients as described by Rupp Howa~-Fianders [16]. Sedimentation was c a n e d out in an SW 50.1 rotor of a Beckman L5-50 centrifuge spun at 30 000 rev.~in for 90 rain at ~ P C Fractions were collected from the top of the gred~nt and the d~st~bu~n of radio. activity throughout the gradient was determined as described ~ w h e ~ [17].
156 Resul~
The -:ist~nce sedimented by DNA in alkali depends on the single strand molecular weight. We interpret increases (decreases) in sedimentation cor~tant as decreases tincre~u~s) in the numbers of single strand breaks. Our basic observation is shown in Fig. 1. The single strand breaks appearing in the D N A of irradiatedwild type cellsare only slightlyrepai~d if the cellsm ~ incubated in the absence of ATP, but are almost completely repaired if the cells are incubated in the presenc~ of A T P (no other cofactors present). The rate of repair is rap.~das is indicated in Fig. 2 and the repair observed in the presence of A T P is inhibited by N M N , an inhibitor of polynucleotide lig~se. These data suggest that the rejoininginvolves ligaseactivityand the data in Fig. 3, showing that there isno rejoiningin polA cellsincubated in the presence of A T P indicate that polymerese I is also necessary for rejoining.The stimulation of rejoining by A T P is not an artifactof an irreversiblebreakdown of D N A in its absence, because A T P may be added up to 5 rain after the start of incubation at 30°C and stillgive just about complete rejoining (Fig. 4). W e have also made the following observations on irradiated wi~i type cells (data not shown). The optimal A T P concentration for strand rejoining in 9.0 rain is 1.3-d).65 raM. At 0.13 m M rejoining is only half completed. The rejoining reaction needs M g 2÷, N A D , the cofactor of polynucleotide ligasehas littleinfluence on break rejoiningin the presence or absence of ATP. W e wanted to find out whether the enhancing action of A T P on strand break repair was specific or was a general property of other ribonucleotide triphos-
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Pig. 1. Alkaline sucrose gradient sedimentation profiles of DI~A from E. c o l i KMBL, 1056 (wild type). A no irradiation, 20 rain incubation: t , irradiation, no ineubat/on; o. inradiation. 20 m i n i n e u b a t i o n , no additions; e, irradiat/on, 20 rnin incubation PlUS ATP (1.3 mM). The molecular weight scale was derived as in ref. 27.
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Fig. 2. Alkaline sucrose gradient sedimentaUon p~omes o~ DNA f~,cm E. co~ KMBL 1056 (wnd type), A11 samples irradiated, POst-irradiation conditions: o, 20 rain, n o addi~ons: ®, 10 r n ~ p t ~ ATP (1.3 ~h~)~ e. 20 m|n PlUS ATP ~.1.3 raM): ~. 20 rain plus NMN (5 mM) plus ATP (1.3 InM).
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Fig. 3. , c J k ~ n e sucrose gradient sedimentaUon profiles o f DNA from E. coli KMBL 178"/polA. s , n o irradiation, 20 rain incubation, n o additionS; ~, inadiation, n o incubation; o, is~adi~tion, 20 mh~ incuba#./on. n o additionS; @4 in'adiatlon. 20 min i n c u b ~ o n plus ATP (1.3 mM). S i m ~ red,tits have been obtained with DNA frow E. coil KMBL 1795 m A .
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Fig. 4, Alkaline ~etose gradient secUmentatic~n profiles of DNA from E. coil KMElr, 1056 (wild type). Post-in'adlation incubation for 20 min~ o, n o additiolm~ e~ ATP (1.3 raM) added 2.5 rain after start of incubation- ~, ATP (1,3 raM) added 5 rain after start o f incubation.
phates. W e found (Fig. 5) that C T P and G T P are as good as AT~ ~,and that U T P does not stimulate at all (data not shown). Hence, the A T P effect is not specific for that t:,qphosphate,Moreover, the other individualdeoxynucleoside triphosphates also stimulate rejoining in the absence of A T P (Fig, 6a). The best rejoining observed is in the presence of allfour dNTPs plus N A D (Fig. 6b).
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F/g, 5, Alkaline sucrose gradient sedimentation p:~flles of DNA from F, e c l i KMBL 1056 (wild type). Post-irradiation ineub~ttion for 20 rain; o, no additians~ o, CTP (1,3 mM); ^, GTP (0.65 raM).
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~s
Fig. 6,(a and b) Alkaline sucrose gradient sedimentation profiles of DNA ft~m E. coli KMBL 1056 (wikl type). Post*Irradiatlon incubation f o l 20 min; o, n o additions: o, dATP (1~0 ~M): o, TTP (130 ~M)~ ~, four dNTPs (33/JM each) plus NAD (3.3 mM): A, four dDNTPs plus NAD plus ATP (1.4 raM),
Discussion Our data indicate that in toluenized wild type cellsthe re~inh~g of strand breaks resulting from X-irradiation is stimulated by A T P or by other NTPs (except U) or dNTPs. Since the stimulatingeffect of A T P isinhibited by N M N (nicotinarnide mononucleotide), polynucleotide ligase is implicated in the joining process as is to be expected. W v interpret the fact that N A D , the cofactor for ligase,stimulatesthe rejoining process only slightlyin the presence or absence of ATP, as indicatingthat there m a y be sufficientendogenous N A D to accomplish rejoining. L o w levelsof endogenous cofactors m a y be important in the rejoining process because the n u m l ~ of breaks to be joined is relatively small, perhaps of the order of 100 per chromosome. The effect of N A D in the E. colt system are different from those observed in toluenized B. subtU~ in the latterN A D is a strong stimulator of rejoining [18]. Polynucleotide ligase is not sufficient by itselfto rejoin breaks since the addition of N A D in the absence of A T F does not lead to rejoining. Another necessary enzyme system seems to be pol.w~erase I since we observe no rejoining in poIA mutants when A T P is added. This inabilityof poIA cellsto rejoin s~ugle strand breaks does not result from the failure of A T P to enter the toluen~ed cells because in polA cells,as in wild tylm.• cells [13], A T P stimulates ~ e production of single strand breaks following ultravioletirradiation. Not m u c h data exist on conditions of strand break rejoining'~nsemi-permeable cells. Repair synthesis, nct accompanied by break rejoining, has bee~ observed in X-i~adiated polA mutants of E. co/i (19). X-zay treatment induces some reduced non-conservative D N A sFnthesis in polymerase I.defieient mutants of B. subtil~s [20] whereas no ~toration of tmmforming ~ was
observed in similar experiments [8]. Extensive ("long patch") polymerase H and III dependent repair replication was found in ultraviolet-~radiated poty-
160 merase I mutants of E. coli [10--12]. But the poIA independent repair synthesis is not followed by a ligation reaction if incision breaks are allowed to accumulate [21]. Thus, D N A polymemse I seems to be the 0nly polymerase able to take part in break rejoining in semi-permeable cellsalthough two types of break repair mechanisms were shown to existin vivo [22]. Toluenized cells possibly loose some factor necessary for break repairby the second mechanism. The stimulatingeffect of A T P or other nucleoside triphosphates for rejoining of breaks is not easy to explain. In X-irradiated cellsA T P does not seem to stimulate an incision step. Even in poIA cellswhere strand rejoining is lacking no more breaks are found in the presence of A T P than in its absence. Thus the stimulating effect of A T P or other triphosphates m a y have to do with the gap filling process it.ll or a process closely related to it, especially since the numbers of gaps to be filledis small and small pools of endogenous factors m a y almost be sufficientto accomplish rejoining. A T P partiallystimulatesthe nonconservative repair type synthesis occurring in 7-irrediatedB. subtilis cells [20] but has no effect on the restoration of the transforming activityand molecular weight of its D N A [7,8]. Studies that measure repair synthesis are not directly comparable with ours since A T P and allfour dNTPs are used in them, but only A T P is needed for extensive strand rejoiningin toluenized wild type cells. The effect of A T P or other ~iphosphates on strand rejoiningafter X-irradia. tion is very different from its effects on ultraviolet-irradiatedpermeabilized cells. In the latter it stimulates the appearance of single strand breaks by a process that seems to be mediated by the uvrA + uvrB gene products [13]. Moreover, A T P is needed for repairsynthesis in ultraviolet.irradiat~_~d poIA cells [10--12]. The stimulatory effects of the various triphosphates for strand rejoining show more analogy to the repair following nuclease treatment of toluenized cells [14]. In this system no repair type synthesis was observed in poIA cells, and A T P was not necessary for repair synthesis in wild type ceils, but, of course, the ot~herdl'PrPs were present to measure such synthesis. The amount of repair replication following ionizing nldiation has not been estimated directly in E. coli. Its amount depends on the endogenous level of ligese [23]. High amounts of ligaselimit the amount of nucleotide insertion.In mammalian cellsthe numbers of nucleotides added per chain break produced is about of 2--3 [24--271. Despite our lack of knowledge about the detailsin the processes observed, our data indicate that there,is no need for extensive repair replication in the strand rejoiningin toluenized cells.Our observations are consistent with the idea that following X-irradiationthe ends of the broken regions must be "tailoredand one or a few nucleotides mlded by polymerase I, followed by ligation. Acknowledgments This investigation was sponsored by the Jewish Agency through the Israeli Center of Absorption in Science. W e are indebted to R. Ben-lshai for supplying bacterial strains and S. Manoilis for perforrning some of the experiments. This work was carried out, in part, at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Develol~ment Administration.
References 1 Set~ow, R,B. and Setlow, J.K. (1972) Ann. Rev. B~ophys. ~ , 1, 2~3--34~; 2 Kapp, D.S. an8 Smith, K.C. (1969) |nt. J. RaciSt, ~Ioi, 14, ~ 7 ~ T | 3 JaeobQ, A,, Bopp, A. ~ d H~[en, U. (t972) In|. J. ~a~.iat. ~ . 22~ 431--43~ 4 I~ndbeck, L. 8,nd H a p n , U, (1973) B l o c h ~ . ~ b y s , A ~ 331, 3 ~ - 4 2 7 ~) L~lpis~ P,J. and Gim¢~,'~, A,F, (1972) Pro~. NaU. A©ad. Sci. U,S. 69, 3=11--3214 fJ Fodo~, I,, Matvlenko, N,|,, Zlot~d~ov. K,M,, T a Q y ~ , V,L, S ~ n , A.S. ~ ~ v A~ {1)7~ Nucleic Acid R ~ , 2, 63r)~646 7 Nogutl, T, and Kada, T. (1972) J. Mo]. Biol. ~?, I"~07--512 8 N o g t l , T. and Kada, T, (197~)) ~OChi~l. Biophy~. Ae([& 3~5, 2 ~ - - ~ 3 9 Matmldalta, H, and I ~ r u n o , I, (I~)72) Biochim, ~ p h y s . A ¢ ~ ~'~2. 2 0 ~ - - 2 | I 19 Masker, W.E, an(1Ha~w~It, P.C, (19~3) Pro~, Na~. A©~d, SoL U,S, ~O, 1 ~ 1 3 3 11 M ~ k c r , W.E,~ H~naw~dt, P,C, and Shizuy~. H, (19#3) Nal, New ~ c ] . ~ 4 , ~ 2 ~ 3 12 Masker, W.E, an0. H a n a w ~ t , P C , (~974) ~. MoL ~ o l , 8 8 , 1 3 - - 2 3 13 Waldstein, E.A., Sharon, 11. and Bert.l~h~, R, (1974) P~o~. N ~ , A e ~ , S~i, ~,S, #I~ ~ b 1 ~ 14 Moses, R,E, ~ I d R i c h a ~ i ~ n , C.C, (19"~0) PI,~, Natl, Ac~l, SoL ~LS. 6~. 6 " ~ 4 - ~ I 15 Billen, D, (1969) ,~, Baeteriol, 9#, 1169.-1175 16 Rupp, W,D, ~nd ~ow&~rd-Fl~ders, P, ( I ~ 8 ) J. M ~ , ~ , 31, ~ I ~ 0 ~ 17 Carrier, W,L and Setlow, R,B. (1971) A~a]. ~ ¢ h ~ , 43, 4 2 # - ~ 3 2 18 B~len, D, and H e l I e r m ~ G,R, (19~'4) |~ochlm. ~ o p h y s . A c ~ 383, 3 7 ~ - - 3 ~ 19 Gazicv, A,J., Sert~eva, S,A, and Kuzin, A,M. (1974) l ~ c . A c ~ . S~L ~LS.S~R., ~ , S~L ~, 49~--497 2 0 Billen~ D. a~.',. Hellerm~nn, G.R, (1974) 3 1 o c h ~ . ~ s . A~a 3~, I~5--I~ 21 Sharon~ R.0 Miller, C, ~md Ben-lsh~, R, (1975) J. B ~ r ~ o l , 123,11~'--111~ 22 Town, C,D., Smith, K,C, and KaI~lan, H,S, (1971) Sc~r~ce 1~2, ~ 5 1 - - ~ 23 B ~ I ~ , D., H e l l e ~ a n n , G,R, ~ d StalUo~, D,R, (19~5) J, B ~ e ~ , I~4, ~ 24 Painler~ R,B, ~ d Yown~, B.~, (1972) M:z~aL Res. 14, 225--235 2 5 Fox, M, and Fox, B,W, (1973) Int, J, Radi~t. Bi~d. 23,333.--~5~ 26 Donl~n, T. and Norman, A, (1971) Mut~t, Re~ 13, 9"~--I07 27 Rel[~n~ 3,D, an0 SeUow, B,~I, ~1974) C ~ c e r Re~. 34, 3318--3325