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Mechanism of the ATP effect in the DNA repair synthesis of γ-irradiated Escherichia coli cells
247
Biochimica et Biophysica Acta, 607 (1980) 247--255 © Elsevier/North-Holland Biomedical Press
BBA 99634 MECHANISM OF THE ATP E F F E C T IN THE DNA R E P A I R SYNTHESIS OF ")'-IRRADIATED E S C H E R I C H I A C O L I CELLS
CHRISTEL G/kRTNER a,,, EVELYN A. WALDSTEIN b and ULRICH HAGEN a,, a Kernforschungszentrum Karlsruhe, Institut fiir Genetik und fiir Toxikologie yon Spaltstoffen, Karlsruhe (F.R.G.) and b Tel Aviv University, Department of Biochemistry, Ramat Aviv (Israel)
(Received July 16th, 1979) Key words: DNA repair synthesis; 7-irradiation; ATP stimulation; Toluene; Permeability; (E. eoli)
Summary Gamma-irradiation of Escherichia coli cells made permeable to deoxynucleoside triphosphates (dNTP) b y toluene induces a repair-type D N A synthesis. As previous studies have shown ATP stimulates this DNA synthesis; we studied the mechanism of the ATP effect b y analyzing the kinetics of nucleotide incorporation at various dNTP concentrations. The V values of the DNA repair synthesis rise with increasing dose (0-50 Gy); nonirradiated cells showed a negligible nucleotide incorporation. The apparent Michaelis constant K M for dNTP in the assay was 83--143/~M and the value was much higher than for a DNA polymerase reaction in vitro. ATP stimulated the DNA synthesis with concomitant decrease of KM y e t unchanged V values. Similar results were obtained with a rec BC strain. We propose that the ATP effect is due to a greater affinity of dNTPs to the DNA polymerase, possibly by a stabilisation of the structural integrity of the complex DNA with repair enzymes. Activation of exonucleases b y ATP could be excluded. Addition of NAD to the reaction mixture inhibits the DNA synthesis possibly b y activation of ligase which closes the nicks in the DNA strand.
Introduction In Escherichia eoli cells made permeable to deoxynucleoside triphosphates (dNTP) non-conservative DNA repair synthesis can be observed after ultra* Present
address: Gesellschaft far Strahlen-und Umweltforschung, I n s t i t u t fox Biologie, Abt. Strahlenbiologie, Ingolst//dter Landstrasse 1, 8042 Neuherberg0 F.R.G.
248
violet irradiation [1--3 ], after ~- and X-irradiation [ 4 - 6 ] or after treatment of the cells with alkylating agents [7--9]. DNA polymerase I is responsible for this repair synthesis [3,4] but also other polymerases can substitute partly for this reaction [1--4,6--15,17,22]. The non-conservative DNA repair synthesis in toluene-treated E. coli cells is stimulated by the addition of ATP [ 1,2,7,11]. For the enzymatic polymerizing reaction itself, however, ATP is not necessary. It was shown [12--14] that after ultraviolet irradiation ATP is necessary for the incision of DNA strandbreaks and this reaction produces the substrate for non-conservative DNA synthesis. After this step, repair replication proceeds at the normal rate in the absence of ATP. The mechanism of ATP stimulation of DNA repair synthesis after ionizing radiation is not well understood. Stabilisation of the precursor pool by ATP [ 11], stimulation of ATP dependent nucleases which may widen the gap in the D NA molecule during the normal exicison process [11,12], activation of other polymerases by ATP [7,12] or a faster rate of polymerisation in the presence of ATP [15] have been suggested. To explain the mechanism of the ATP effect on DNA repair synthesis, dNTP incorporation was studied in v-irradiated toluene-treated E. coli cells at various dNTP and ATP concentrations in the assay mixture. The enzyme kinetics of DNA polymerisation in this semi-in-vivo system were analyzed by LineweaverBurk-plots. The involvement of exonucleases in repair synthesis was investigated by the use of the rec BC- mutant, defective in the ATP dependent exonuclease V. The contribution of the rec A gene product to the ATP effect on DNA repair synthesis was also investigated by testing the strains AB 2463 rec A 13- and the isogenic non-mutated strain AB 1157, carrying the rec A ÷ allele.
Materials and Methods The experiments were performed with the E. coli strains JC 4583 his- end I- rec BC÷ and JC 4584 his- end I- rec B21C~2 deficient in exonuclease V [16] and AB 1157 (thr, leu, pro, his, arg, thi, lac, ara, xyl, mtl, gal, ts x, str, sup E, )~-) and the isogenic AB 2463 rec A 13- mutant. The strains were grown in minimal medium M9 supplemented with histidine (5 #g/ml), vitamin B-1 (1 ~g/ml) and casamino acids (0.5%, pH 7). Exponentially growing cells were harvested by centrifugation (10 min at 104 rev./min, Sorvall Superspeed RC-2B, Rotor SM 24) at a density of 2.5. l 0 s cells/ml, washed twice with 0.05 M potassium phosphate buffer, pH 7 . 4 . 2 . 5 . 1 0 9 cells/ml, 10-fold concentrated, were gently stirred (Vortex) with 1% toluene in phosphate buffer for 3 rain at room temperature, diluted 10-fold and washed twice again with phosphate buffer. During irradiation in ice (6°Co~7-source, Atomic Energy of Canada Ltd., dose rate 2.15 Gy/min, respectively, 53 Gy/min) the cell suspension had a final concentration of 5 . 1 0 9 cells/ml. 50-#1 aliquots ( 2 . 5 . 1 0 s cells) were mixed with a 50 #1 incubation mixture at a final concentration of 0.07 M phosphate buffer, 0.01 M MgC12 and 2 #Ci ~ 0.1 ~M. [3H]Thymidine-5'-triphosphate (30 Ci/mmol, Amersham-Buchler) was used as radioactive precursor. In addition the incubation mixture contained various amounts of ATP (0--2 mM, Serva),
249
NAD (1 mM, Boehringer) and deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP, 0--20 /~M each, Serva) as indicated. The incubation period was for 30 min at 37°C. This time was chosen because in preliminary experiments it reflected a linear rise of dNTP incorporation with time. The reaction was stopped in ice and 3 ml of ice-cold 10% trichloroacetic acid/2% sodium pyrophosphate solution were added. After 15 min on ice the acid-insoluble DNA was collected on a Sartorius membrane filter, pore size 0.45 #, washed three times with 4 ml 5% trichloroacetic acid/l% sodium pyrophosphate and twice with 4 ml 0.01 M HC1. The filters were dried and counted in 10 ml toluenePPO-POPOP-scintillation fluid in a Packard Tri-Carb Spectrometer. Results and Discussion Incubation of toluene-treated E. coli cells in the presence of all four dNTPs results in incorporation of dNMPs. We have found that this incorporation is low in nonirradiated cells, as shown in Fig. 1 and that it increases with the dose of irradiation. The number of incorporated dNMPs in wild type cells (JC 4583) was small when only 2 ~M of each dNTP were present as compared to a concentration of 20 #M of each dNTP. At the latter concentration the incorporation increased linearly with the dose up to 50 Gy. The results suggest that the DNA synthesis observed reflects a repair-type reaction. In the presence of
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Radiotion Dose | G y l F i g . 1. D N A r e p a i r s y n t h e s i s in ~/-irradiated E. coli cells. (e ~) E. coli s t r a i n J C 4 5 8 3 h i s - a n d I r e c BC + i n c u b a t e d f o r 3 0 m i n w i t h 2 0 9 M o f e a c h d N T P ; ( o o) w i t h 2~uM o f e a c h d N T P ; (A A) E. coli s t r a i n J C 4 5 8 4 h i s - e n d I - t e e B 2 1 C ~ 2 i n c u b a t e d f o r 3 0 r a i n w i t h 2 0 /~M o f each d N T P a n d 2 / ~ M [ 3 H ] d T T P per test t u b e .
250
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dTTP-coneentration [~M] Fig. 2. I s o t o p e d i l u t i o n e x p e r i m e n t o n D N A r e p a i r s y n t h e s i s . I r r a d i a t e d ( 2 5 G y ) E. coli rec BC + cells w e r e i n c u b a t e d w i t h 2 # M (e e ) o r 1 0 # M (o o) o f e a c h d A T P , d C T P , d G T P a n d i n c r e a s i n g d T T P c o n c e n t r a t i o n s . T h e c o n c e n t r a t i o n o f [ 3 H ] d T T P w a s 0 . 1 /aM ~- 2 C i / 1 0 0 1 r e a c t i o n m i x t u r e .
20 /~M of each dNTP the rec BC- cells incorporated much less reaching approx. 15% nucleotide incorporation as compared to that in wild type cells (Fig. 1). Toluene treatment renders cells permeable for small molecules like dNTP, ATP or NAD [17] but one cannot be sure that these compounds are completely removed from the cells during the washing procedure. To test this experimentally the internal pool of dNTPs after toluene treatment was evaluated by isotope dilution experiments (Fig. 2). Irradiated cells were incubated with a constant amount of [3H]dTTP (0.1 /~M), 2 or 10 uM of dATP, dCTP and dGTP together with increasing amounts of unlabeled dTTP. The results when plotted as reciprocal incorporated radioactivity against the concentration of dTTP show that the incorporated [3H]dTTP was diluted into newly synthesized DNA proportionally to the concentration of unlabeled dTTP. The extrapolation to zero exhibits a value of 1.6 ~M dTTP which can be seen as a residual internal pool. This amount is small in comparison to the added concentrations of dNTPs (up to 20 ~M each) used for kinetic studies. We assume a similar low yield for the residue of the other dNTPs. The optimal ATP concentrations for the kinetic studies were obtained from other preliminary experiments. Irradiated wild type cells (JC 4583) were incubated in the presence of increasing ATP concentrations (Fig. 3). ATP stimulated the incorporation of dNMPs strongly in a concentration range of 0.2-0.5 mM ATP. A plateau was observed in the range of 0.5--1.5 mM ATP with 4 uJVIof each dNTP present (Fig. 3). For rec BC- cells irradiated with 50 Gy we found an optimal stimulation of incorporation with 0.5 mM ATP (Fig. 3).
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ATP concentration [mM] Fig. 3. A T P e f f e c t o n r a d i a t i o n - i n d u c e d DNA repair synthesis. E. coli s t r a i n JC 4 5 8 3 i r r a d i a t e d w i t h I 0 G y ($ -'), 25 G y (o o), 50 G y ( a A), 75 G y (A ~) a n d i n c u b a t e d f o r 30 rain a t 37VC w i t h 4 juM o f e a c h d N T P a n d 0 - - 2 m M A T P . T h e s t r a i n 4 5 8 4 rec B C - was i r r a d i a t e d w i t h 1 0 G y (u . . . . . . B) o r 50 G y (D. . . . . . D) a n d i n c u b a t e d under the same experimental conditions.
When 8 ~M of each dNTP were present in the incubation mixture, smaller concentrations (0.2--0.5 mM) of ATP showed maximal stimulation b u t concentrations of > 0 . 5 mM ATP considerably inhibited the incorporation of dNMPs (data n o t shown). Generally the experiments on DNA synthesis were performed in the absence o f NAD. But in the presence of 1 mM NAD and increasing dNTP concentrations the KM value remained unchanged when plotted according to LineweaverBurk (Table I). We conclude that NAD reduces the nucleotide incorporation to 20--30% as compared to the control b u t that the affinity of dNTPs to the polymerase remains the same. As NAD is an obligatory cofactor for DNA ligase [4], it seems conceivable that in the presence of NAD, ligase seals the nicks and prevents further nucleotide incorporation b y DNA polymerase. For the analysis of the ATP effect on the kinetics of nucleotide incorporation therefore we omitted the NAD in the reaction mixture. DNA polymerases require a 3'-OH-end-group as primer and dNTPs as precursors for chain elongation [18]. Thus the kinetics of the polymerisation can be described as a bisubstrate reaction [19]. By varying either the a m o u n t o f end-groups b y irradiating the cells with increasing 7-ray doses or the a m o u n t
252 TABLE I K M * AND V** VALUES T R E A T E D E. COLI C E L L S
OF THE RADIATION-INDUCED
Strain
REPAIR
SYNTHESIS
IN T O L U E N E -
Radiation dose (Gy) 10
25
50
JC 4 5 8 3 V(±ATP) KM(--ATP) KM(+ATP) R a t i o KM(_ATP)/KM(+ATP ) V(+NAD)
0.3 83 27 3.1 0.2
1.0 111 35 3.2
3.7 143 40 3.6
JC 4 5 8 4 V(_+ A T P ) KM(--ATP) KM(+ATP) R a t i o KM(_ATP)/KM(+ATP )
0.2 300 300 1
0.3 220 118 1.9
1.0 154 87 1.8
* K M i n [~tM d N T P ] . ** V in [ n m o l d N M P i n c o r p ~ . ml • min
o f the precursors b y increasing the dNTP concentration, the kinetics of DNA repair synthesis can be studied. Plotting the data according to Lineweaver-Burk (examples are given in Fig. 4) the apparent values for V and K M in this semi-in-vivo system can be obtained. The results are summarized in Table I. The V values increased with the radiation dose indicating that more substrate is available for repair synthesis at higher doses. Obviously the a m o u n t of radiation damage corresponds to the a m o u n t of starting points for repair synthesis. As illustrated in Fig. 4, addition o f ATP has no influence on the V values, although it enhances the rate of repair synthesis at each dNTP concentration distinctly. Hence in Table I the V values are presented for the presence and absence of ATP. We observed KM values increasing with dose in rec BC ÷ cells. As the K M values reflect the affinity of dNTP to the DNA polymerase, we conclude that higher radiation doses lower this affinity. On the contrary the addition of ATP increases the affinity b y a factor of three. The kinetics of repair synthesis in rec BC- cells showed a different pattern. The repair synthesis in the rec BC- m u t a n t is much lower than in rec BC ÷ cells (Fig. 3) and the observed higher KM values reflect also a lower affinity of dNTPs to the DNA polymerase. ATP has a stimulating effect on the nucleotide incorporation after 25 a n d 50 Gy represented b y lower K ~ values. The rec A ÷ protein catalyzes homologous pair~.g o f superheli~al D N A with single-stranded fragments in an ATP-dependent reaction [20]. If the rec A protein is involved in repair we w o u l d expect a lower incorporation o f dNTPs into DNA of a rec A- mutant. This was n o t found when we tested the E. coli strains AB 1157 rec A ÷ and the isogenic m u t a n t AB 2463 rec A13-. Incorporation of dNTPs was stimulated b y ATP in b o t h strain~ (Table II). Our data lead to the following considerations for the role of ATP in DNA repair synthesis in toluene-treated E. coli cells:
253
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Fig. 4. A T P e f f e c t o n t h e k i n e t i c s o f D N A r e p a i r s y n t h e s i s i n t o l u e n e - t r e a t e d E. c o l i c e l l s . L i n e w e a v e r B u r k - p l o t . S t r a i n J C 4 5 8 3 r e c BC + i r r a d i a t e d w i t h 1 0 G y (A), 2 5 G y (B) o r 5 0 G y (C) a n d i n c u b a t e d w i t h 0 - - 2 0 /JM o f e a c h d N T P w i t h o u t A T P (o o), w i t h 1 m M A T P (e ~) or 0.5 mM ATP (A A). S t r a i n J C 4 5 8 4 r e c B C - i r r a d i a t e d w i t h 2 5 G y (D) a n d 5 0 G y (E) a n d i n c u b a t e d w i t h 0 - - 2 0 ?zM o f e a c h d N T P w i t h o u t A T P (o o), w i t h 1 m M A T P (e -'), r e s p e c t i v e l y 0 . 5 m M (A A).
(i) The amount of primer sites available for the polymerisation reaction is not influenced by ATP as can be concluded from the constant V values. It means that neither the exonuclease V nor any other ATP dependent endo- or exonucleolytic activity is stimulated to produce new primer sites. This corresponds with the finding that in the presence of dNTPs wild type and rec BCceils seal similar fractions of strand breaks. Furthermore ATP does not stimulate the break rejoining activity in rec BC ÷ cells [21]. The experimental results with the rec A- mutant allow us to conclude that the ATP stimulation on the repair synthesis is not due to a stimulation of the rec A protein. (ii) The finding that ATP had no effect on the V values suggests that only one polymerizing enzyme performed 7-dependent repair synthesis in the absence and presence of ATP. Since DNA polymerase II and III are ATP dependent [3], we assume that DNA polymerase I is responsible for the measured repair synthesis. This corresponds with data of Billen and Hellermann
254
T A B L E II R E L A T I O N B E T W E E N I N C O R P O R A T E D [ 3 H ] d T T P ( c p m ) IN T H E P R E S E N C E A N D A B S E N C E OF A T P IN T O L U E N E - T R E A T E D E. C O L I C E L L S , AB 1 1 5 7 W I L D T Y P E A N D AB 2 4 6 3 rec A Strain
R a d i a t i o n dose (Gy) Control
10
25
50
75
100
AB 1 1 5 7 wild t y p e
2 *
2
2.5
2.5 a
2.4
2.6
AB 2463 rec A -
1.5
1.5
2.3
1.9 b
1.9
1.7
* R a t i o c p m + A T P / c p m - - A T P f o r e x a m p l e a 58 2 0 0 b 55 4 9 8 23619
28968
[7] w h o found only a negligible a m o u n t of repair synthesis in pol A- cells even in the presence o f ATP. (iii) The observed stimulation of repair synthesis by ATP is expressed by a lower apparent KM value for the dNTPs. We conclude from this observation that ATP increases the affinity of the precursors to the enzyme-DNA-complex in the cell. Furthermore it should be noted that these apparent KM values in this semi-in-vivo system are much higher than the K M values for dNTPs in vitro for D N A polymerase I acting on nicked DNA which are ranging between 2 and 5/~M [19]. In the cell there is apparently a sterical hindrance which prevents dNTP from acting on the complex between irradiated DNA and repair enzymes, for example D N A polymerase. Thus the effect of ATP could be due to the preservation of the structural integrity of the genome which enhances the availability of dNTP for the enzyme. I n the rec BC- cells the KM values for dNTPs are much higher than in wild t y p e cells indicating a lower affinity of dNTPs to the polymerase. Therefore also the a m o u n t of repair synthesis is much lower (Fig. 3). There is no apparent explanation for this fact except that rec BC- cells in general are more sensitive and much less viable. Toluene treatment could attack the structural integrity of the genome more than in BC ÷ cells. However ATP lead to the same effect as in wild type, i.e. it enhances the affinity of dNTPs to the enzyme, b u t the effect is lower (Table I). Furthermore, like in BC ÷ cells, ATP did n o t change the V values indicating no rise in the n u m b e r of end-groups available for repair synthesis. O u r experiments suggest that cells treated with toluene are a suitable system for kinetic studies on enzymatic reactions involved in DNA repair. Toluenetreated cells at least partly preserve the integrity of the enzyme complexes and thus m a y be preferred to mere in vitro systems although the loss of some cofactors has to be considered. References 1 Maske~, W.E. a n d H a n a w a i t , P.C. ( 1 9 7 3 ) Proc. Natl. A c a d . Sci. U.S.A. 70, 1 2 9 - - 1 3 3 2 M u k a r , W.E. a n d H a n a w a i t , P.C. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 0 , 2 2 9 - - 2 3 6 3 Ben-Ishai, R. a n d S h a r o n , R. ( 1 9 7 7 ) J. Mol. Biol. 1 2 0 , 4 2 3 - - 4 3 2
255
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Billen, D. and Hellermann, G.R. (1976) J. Bacteriol. 120, 785--793 Bfllen, D.. Henermann, G.R. and Stallions, D.R. (1975) J. Bacteriol. 124, 585--588 Billen, D. and Hellermann, G.R. (1977) Int. J. Radiat. Biol. 3 1 , 3 8 5 - - 3 8 9 Billen, D. and Hellermann, G.R. (1977) Chem. Biol. Interactions 18, 365--380 Thiehnann, H.W., Vosberg, H.P. and Reygers, U. (1975) EULr.J. Biochem. 5 6 , 4 3 3 - - 4 4 7 Thielmann, H.W. (1976) Ettr. J. Biochem. 6 1 , 5 0 1 - - 5 1 3 Masker, W.E. and Hanawalt, P.C. (1973) Nature New Biol. 244, 242--243 Masker, W.E. and Hanawalt, P.C. (1974) J. Mol. Biol. 88, 13--23 Masker, W.E. (1976) Biochim. Biophys. Acta 442, 162--173 Waldstein, E.A., Sharon, R. and Ben-Ishai, R. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2 6 5 1 - - 2 6 5 4 Dorson, J.W. and Moses, R.E. (1978) J. Biol. Chem. 2 5 3 , 6 6 5 - - 6 7 0 Billen, D. and Hellen~nann, G.R. (1974) Biochim. Biophys. Acta 3 6 1 , 1 6 6 - - 1 7 5 Clark, A.J. (1973) Ann. Rev. Genet. 7, 67--86 Moses. R.E. and Richardson, C.C. (1970) P~oc. Natl. Acad. Sci. U.S.A. 6 7 , 6 7 4 - - 6 8 1 Landbeck, L. and Hagen, U. (1973) Biochim. Biophys. Acta 3 3 1 , 3 1 8 - - 3 2 7 B~hner, R. and Hagen, U. (1977) Biochim. Biophys. Acta 479, 300--310 Weinstock, G.M., McEntee, K. and Lehman. I.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 7 6 , 1 2 6 - - 1 3 0 Waldstein, E.A. (1979) J. BacterioL 1 3 9 . 1 - - 7 Waldstein, E.A. and Setlow, R.B. (1976) Biochim. Biophys. Acta 442, 154--161
Biochimica et Biophysica Acta, 607 (1980) 247--255 © Elsevier/North-Holland Biomedical Press
BBA 99634 MECHANISM OF THE ATP E F F E C T IN THE DNA R E P A I R SYNTHESIS OF ")'-IRRADIATED E S C H E R I C H I A C O L I CELLS
CHRISTEL G/kRTNER a,,, EVELYN A. WALDSTEIN b and ULRICH HAGEN a,, a Kernforschungszentrum Karlsruhe, Institut fiir Genetik und fiir Toxikologie yon Spaltstoffen, Karlsruhe (F.R.G.) and b Tel Aviv University, Department of Biochemistry, Ramat Aviv (Israel)
(Received July 16th, 1979) Key words: DNA repair synthesis; 7-irradiation; ATP stimulation; Toluene; Permeability; (E. eoli)
Summary Gamma-irradiation of Escherichia coli cells made permeable to deoxynucleoside triphosphates (dNTP) b y toluene induces a repair-type D N A synthesis. As previous studies have shown ATP stimulates this DNA synthesis; we studied the mechanism of the ATP effect b y analyzing the kinetics of nucleotide incorporation at various dNTP concentrations. The V values of the DNA repair synthesis rise with increasing dose (0-50 Gy); nonirradiated cells showed a negligible nucleotide incorporation. The apparent Michaelis constant K M for dNTP in the assay was 83--143/~M and the value was much higher than for a DNA polymerase reaction in vitro. ATP stimulated the DNA synthesis with concomitant decrease of KM y e t unchanged V values. Similar results were obtained with a rec BC strain. We propose that the ATP effect is due to a greater affinity of dNTPs to the DNA polymerase, possibly by a stabilisation of the structural integrity of the complex DNA with repair enzymes. Activation of exonucleases b y ATP could be excluded. Addition of NAD to the reaction mixture inhibits the DNA synthesis possibly b y activation of ligase which closes the nicks in the DNA strand.
Introduction In Escherichia eoli cells made permeable to deoxynucleoside triphosphates (dNTP) non-conservative DNA repair synthesis can be observed after ultra* Present
address: Gesellschaft far Strahlen-und Umweltforschung, I n s t i t u t fox Biologie, Abt. Strahlenbiologie, Ingolst//dter Landstrasse 1, 8042 Neuherberg0 F.R.G.
248
violet irradiation [1--3 ], after ~- and X-irradiation [ 4 - 6 ] or after treatment of the cells with alkylating agents [7--9]. DNA polymerase I is responsible for this repair synthesis [3,4] but also other polymerases can substitute partly for this reaction [1--4,6--15,17,22]. The non-conservative DNA repair synthesis in toluene-treated E. coli cells is stimulated by the addition of ATP [ 1,2,7,11]. For the enzymatic polymerizing reaction itself, however, ATP is not necessary. It was shown [12--14] that after ultraviolet irradiation ATP is necessary for the incision of DNA strandbreaks and this reaction produces the substrate for non-conservative DNA synthesis. After this step, repair replication proceeds at the normal rate in the absence of ATP. The mechanism of ATP stimulation of DNA repair synthesis after ionizing radiation is not well understood. Stabilisation of the precursor pool by ATP [ 11], stimulation of ATP dependent nucleases which may widen the gap in the D NA molecule during the normal exicison process [11,12], activation of other polymerases by ATP [7,12] or a faster rate of polymerisation in the presence of ATP [15] have been suggested. To explain the mechanism of the ATP effect on DNA repair synthesis, dNTP incorporation was studied in v-irradiated toluene-treated E. coli cells at various dNTP and ATP concentrations in the assay mixture. The enzyme kinetics of DNA polymerisation in this semi-in-vivo system were analyzed by LineweaverBurk-plots. The involvement of exonucleases in repair synthesis was investigated by the use of the rec BC- mutant, defective in the ATP dependent exonuclease V. The contribution of the rec A gene product to the ATP effect on DNA repair synthesis was also investigated by testing the strains AB 2463 rec A 13- and the isogenic non-mutated strain AB 1157, carrying the rec A ÷ allele.
Materials and Methods The experiments were performed with the E. coli strains JC 4583 his- end I- rec BC÷ and JC 4584 his- end I- rec B21C~2 deficient in exonuclease V [16] and AB 1157 (thr, leu, pro, his, arg, thi, lac, ara, xyl, mtl, gal, ts x, str, sup E, )~-) and the isogenic AB 2463 rec A 13- mutant. The strains were grown in minimal medium M9 supplemented with histidine (5 #g/ml), vitamin B-1 (1 ~g/ml) and casamino acids (0.5%, pH 7). Exponentially growing cells were harvested by centrifugation (10 min at 104 rev./min, Sorvall Superspeed RC-2B, Rotor SM 24) at a density of 2.5. l 0 s cells/ml, washed twice with 0.05 M potassium phosphate buffer, pH 7 . 4 . 2 . 5 . 1 0 9 cells/ml, 10-fold concentrated, were gently stirred (Vortex) with 1% toluene in phosphate buffer for 3 rain at room temperature, diluted 10-fold and washed twice again with phosphate buffer. During irradiation in ice (6°Co~7-source, Atomic Energy of Canada Ltd., dose rate 2.15 Gy/min, respectively, 53 Gy/min) the cell suspension had a final concentration of 5 . 1 0 9 cells/ml. 50-#1 aliquots ( 2 . 5 . 1 0 s cells) were mixed with a 50 #1 incubation mixture at a final concentration of 0.07 M phosphate buffer, 0.01 M MgC12 and 2 #Ci ~ 0.1 ~M. [3H]Thymidine-5'-triphosphate (30 Ci/mmol, Amersham-Buchler) was used as radioactive precursor. In addition the incubation mixture contained various amounts of ATP (0--2 mM, Serva),
249
NAD (1 mM, Boehringer) and deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP, 0--20 /~M each, Serva) as indicated. The incubation period was for 30 min at 37°C. This time was chosen because in preliminary experiments it reflected a linear rise of dNTP incorporation with time. The reaction was stopped in ice and 3 ml of ice-cold 10% trichloroacetic acid/2% sodium pyrophosphate solution were added. After 15 min on ice the acid-insoluble DNA was collected on a Sartorius membrane filter, pore size 0.45 #, washed three times with 4 ml 5% trichloroacetic acid/l% sodium pyrophosphate and twice with 4 ml 0.01 M HC1. The filters were dried and counted in 10 ml toluenePPO-POPOP-scintillation fluid in a Packard Tri-Carb Spectrometer. Results and Discussion Incubation of toluene-treated E. coli cells in the presence of all four dNTPs results in incorporation of dNMPs. We have found that this incorporation is low in nonirradiated cells, as shown in Fig. 1 and that it increases with the dose of irradiation. The number of incorporated dNMPs in wild type cells (JC 4583) was small when only 2 ~M of each dNTP were present as compared to a concentration of 20 #M of each dNTP. At the latter concentration the incorporation increased linearly with the dose up to 50 Gy. The results suggest that the DNA synthesis observed reflects a repair-type reaction. In the presence of
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Radiotion Dose | G y l F i g . 1. D N A r e p a i r s y n t h e s i s in ~/-irradiated E. coli cells. (e ~) E. coli s t r a i n J C 4 5 8 3 h i s - a n d I r e c BC + i n c u b a t e d f o r 3 0 m i n w i t h 2 0 9 M o f e a c h d N T P ; ( o o) w i t h 2~uM o f e a c h d N T P ; (A A) E. coli s t r a i n J C 4 5 8 4 h i s - e n d I - t e e B 2 1 C ~ 2 i n c u b a t e d f o r 3 0 r a i n w i t h 2 0 /~M o f each d N T P a n d 2 / ~ M [ 3 H ] d T T P per test t u b e .
250
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dTTP-coneentration [~M] Fig. 2. I s o t o p e d i l u t i o n e x p e r i m e n t o n D N A r e p a i r s y n t h e s i s . I r r a d i a t e d ( 2 5 G y ) E. coli rec BC + cells w e r e i n c u b a t e d w i t h 2 # M (e e ) o r 1 0 # M (o o) o f e a c h d A T P , d C T P , d G T P a n d i n c r e a s i n g d T T P c o n c e n t r a t i o n s . T h e c o n c e n t r a t i o n o f [ 3 H ] d T T P w a s 0 . 1 /aM ~- 2 C i / 1 0 0 1 r e a c t i o n m i x t u r e .
20 /~M of each dNTP the rec BC- cells incorporated much less reaching approx. 15% nucleotide incorporation as compared to that in wild type cells (Fig. 1). Toluene treatment renders cells permeable for small molecules like dNTP, ATP or NAD [17] but one cannot be sure that these compounds are completely removed from the cells during the washing procedure. To test this experimentally the internal pool of dNTPs after toluene treatment was evaluated by isotope dilution experiments (Fig. 2). Irradiated cells were incubated with a constant amount of [3H]dTTP (0.1 /~M), 2 or 10 uM of dATP, dCTP and dGTP together with increasing amounts of unlabeled dTTP. The results when plotted as reciprocal incorporated radioactivity against the concentration of dTTP show that the incorporated [3H]dTTP was diluted into newly synthesized DNA proportionally to the concentration of unlabeled dTTP. The extrapolation to zero exhibits a value of 1.6 ~M dTTP which can be seen as a residual internal pool. This amount is small in comparison to the added concentrations of dNTPs (up to 20 ~M each) used for kinetic studies. We assume a similar low yield for the residue of the other dNTPs. The optimal ATP concentrations for the kinetic studies were obtained from other preliminary experiments. Irradiated wild type cells (JC 4583) were incubated in the presence of increasing ATP concentrations (Fig. 3). ATP stimulated the incorporation of dNMPs strongly in a concentration range of 0.2-0.5 mM ATP. A plateau was observed in the range of 0.5--1.5 mM ATP with 4 uJVIof each dNTP present (Fig. 3). For rec BC- cells irradiated with 50 Gy we found an optimal stimulation of incorporation with 0.5 mM ATP (Fig. 3).
251
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When 8 ~M of each dNTP were present in the incubation mixture, smaller concentrations (0.2--0.5 mM) of ATP showed maximal stimulation b u t concentrations of > 0 . 5 mM ATP considerably inhibited the incorporation of dNMPs (data n o t shown). Generally the experiments on DNA synthesis were performed in the absence o f NAD. But in the presence of 1 mM NAD and increasing dNTP concentrations the KM value remained unchanged when plotted according to LineweaverBurk (Table I). We conclude that NAD reduces the nucleotide incorporation to 20--30% as compared to the control b u t that the affinity of dNTPs to the polymerase remains the same. As NAD is an obligatory cofactor for DNA ligase [4], it seems conceivable that in the presence of NAD, ligase seals the nicks and prevents further nucleotide incorporation b y DNA polymerase. For the analysis of the ATP effect on the kinetics of nucleotide incorporation therefore we omitted the NAD in the reaction mixture. DNA polymerases require a 3'-OH-end-group as primer and dNTPs as precursors for chain elongation [18]. Thus the kinetics of the polymerisation can be described as a bisubstrate reaction [19]. By varying either the a m o u n t o f end-groups b y irradiating the cells with increasing 7-ray doses or the a m o u n t
252 TABLE I K M * AND V** VALUES T R E A T E D E. COLI C E L L S
OF THE RADIATION-INDUCED
Strain
REPAIR
SYNTHESIS
IN T O L U E N E -
Radiation dose (Gy) 10
25
50
JC 4 5 8 3 V(±ATP) KM(--ATP) KM(+ATP) R a t i o KM(_ATP)/KM(+ATP ) V(+NAD)
0.3 83 27 3.1 0.2
1.0 111 35 3.2
3.7 143 40 3.6
JC 4 5 8 4 V(_+ A T P ) KM(--ATP) KM(+ATP) R a t i o KM(_ATP)/KM(+ATP )
0.2 300 300 1
0.3 220 118 1.9
1.0 154 87 1.8
* K M i n [~tM d N T P ] . ** V in [ n m o l d N M P i n c o r p ~ . ml • min
o f the precursors b y increasing the dNTP concentration, the kinetics of DNA repair synthesis can be studied. Plotting the data according to Lineweaver-Burk (examples are given in Fig. 4) the apparent values for V and K M in this semi-in-vivo system can be obtained. The results are summarized in Table I. The V values increased with the radiation dose indicating that more substrate is available for repair synthesis at higher doses. Obviously the a m o u n t of radiation damage corresponds to the a m o u n t of starting points for repair synthesis. As illustrated in Fig. 4, addition o f ATP has no influence on the V values, although it enhances the rate of repair synthesis at each dNTP concentration distinctly. Hence in Table I the V values are presented for the presence and absence of ATP. We observed KM values increasing with dose in rec BC ÷ cells. As the K M values reflect the affinity of dNTP to the DNA polymerase, we conclude that higher radiation doses lower this affinity. On the contrary the addition of ATP increases the affinity b y a factor of three. The kinetics of repair synthesis in rec BC- cells showed a different pattern. The repair synthesis in the rec BC- m u t a n t is much lower than in rec BC ÷ cells (Fig. 3) and the observed higher KM values reflect also a lower affinity of dNTPs to the DNA polymerase. ATP has a stimulating effect on the nucleotide incorporation after 25 a n d 50 Gy represented b y lower K ~ values. The rec A ÷ protein catalyzes homologous pair~.g o f superheli~al D N A with single-stranded fragments in an ATP-dependent reaction [20]. If the rec A protein is involved in repair we w o u l d expect a lower incorporation o f dNTPs into DNA of a rec A- mutant. This was n o t found when we tested the E. coli strains AB 1157 rec A ÷ and the isogenic m u t a n t AB 2463 rec A13-. Incorporation of dNTPs was stimulated b y ATP in b o t h strain~ (Table II). Our data lead to the following considerations for the role of ATP in DNA repair synthesis in toluene-treated E. coli cells:
253
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Fig. 4. A T P e f f e c t o n t h e k i n e t i c s o f D N A r e p a i r s y n t h e s i s i n t o l u e n e - t r e a t e d E. c o l i c e l l s . L i n e w e a v e r B u r k - p l o t . S t r a i n J C 4 5 8 3 r e c BC + i r r a d i a t e d w i t h 1 0 G y (A), 2 5 G y (B) o r 5 0 G y (C) a n d i n c u b a t e d w i t h 0 - - 2 0 /JM o f e a c h d N T P w i t h o u t A T P (o o), w i t h 1 m M A T P (e ~) or 0.5 mM ATP (A A). S t r a i n J C 4 5 8 4 r e c B C - i r r a d i a t e d w i t h 2 5 G y (D) a n d 5 0 G y (E) a n d i n c u b a t e d w i t h 0 - - 2 0 ?zM o f e a c h d N T P w i t h o u t A T P (o o), w i t h 1 m M A T P (e -'), r e s p e c t i v e l y 0 . 5 m M (A A).
(i) The amount of primer sites available for the polymerisation reaction is not influenced by ATP as can be concluded from the constant V values. It means that neither the exonuclease V nor any other ATP dependent endo- or exonucleolytic activity is stimulated to produce new primer sites. This corresponds with the finding that in the presence of dNTPs wild type and rec BCceils seal similar fractions of strand breaks. Furthermore ATP does not stimulate the break rejoining activity in rec BC ÷ cells [21]. The experimental results with the rec A- mutant allow us to conclude that the ATP stimulation on the repair synthesis is not due to a stimulation of the rec A protein. (ii) The finding that ATP had no effect on the V values suggests that only one polymerizing enzyme performed 7-dependent repair synthesis in the absence and presence of ATP. Since DNA polymerase II and III are ATP dependent [3], we assume that DNA polymerase I is responsible for the measured repair synthesis. This corresponds with data of Billen and Hellermann
254
T A B L E II R E L A T I O N B E T W E E N I N C O R P O R A T E D [ 3 H ] d T T P ( c p m ) IN T H E P R E S E N C E A N D A B S E N C E OF A T P IN T O L U E N E - T R E A T E D E. C O L I C E L L S , AB 1 1 5 7 W I L D T Y P E A N D AB 2 4 6 3 rec A Strain
R a d i a t i o n dose (Gy) Control
10
25
50
75
100
AB 1 1 5 7 wild t y p e
2 *
2
2.5
2.5 a
2.4
2.6
AB 2463 rec A -
1.5
1.5
2.3
1.9 b
1.9
1.7
* R a t i o c p m + A T P / c p m - - A T P f o r e x a m p l e a 58 2 0 0 b 55 4 9 8 23619
28968
[7] w h o found only a negligible a m o u n t of repair synthesis in pol A- cells even in the presence o f ATP. (iii) The observed stimulation of repair synthesis by ATP is expressed by a lower apparent KM value for the dNTPs. We conclude from this observation that ATP increases the affinity of the precursors to the enzyme-DNA-complex in the cell. Furthermore it should be noted that these apparent KM values in this semi-in-vivo system are much higher than the K M values for dNTPs in vitro for D N A polymerase I acting on nicked DNA which are ranging between 2 and 5/~M [19]. In the cell there is apparently a sterical hindrance which prevents dNTP from acting on the complex between irradiated DNA and repair enzymes, for example D N A polymerase. Thus the effect of ATP could be due to the preservation of the structural integrity of the genome which enhances the availability of dNTP for the enzyme. I n the rec BC- cells the KM values for dNTPs are much higher than in wild t y p e cells indicating a lower affinity of dNTPs to the polymerase. Therefore also the a m o u n t of repair synthesis is much lower (Fig. 3). There is no apparent explanation for this fact except that rec BC- cells in general are more sensitive and much less viable. Toluene treatment could attack the structural integrity of the genome more than in BC ÷ cells. However ATP lead to the same effect as in wild type, i.e. it enhances the affinity of dNTPs to the enzyme, b u t the effect is lower (Table I). Furthermore, like in BC ÷ cells, ATP did n o t change the V values indicating no rise in the n u m b e r of end-groups available for repair synthesis. O u r experiments suggest that cells treated with toluene are a suitable system for kinetic studies on enzymatic reactions involved in DNA repair. Toluenetreated cells at least partly preserve the integrity of the enzyme complexes and thus m a y be preferred to mere in vitro systems although the loss of some cofactors has to be considered. References 1 Maske~, W.E. a n d H a n a w a i t , P.C. ( 1 9 7 3 ) Proc. Natl. A c a d . Sci. U.S.A. 70, 1 2 9 - - 1 3 3 2 M u k a r , W.E. a n d H a n a w a i t , P.C. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 0 , 2 2 9 - - 2 3 6 3 Ben-Ishai, R. a n d S h a r o n , R. ( 1 9 7 7 ) J. Mol. Biol. 1 2 0 , 4 2 3 - - 4 3 2
255
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Billen, D. and Hellermann, G.R. (1976) J. Bacteriol. 120, 785--793 Bfllen, D.. Henermann, G.R. and Stallions, D.R. (1975) J. Bacteriol. 124, 585--588 Billen, D. and Hellermann, G.R. (1977) Int. J. Radiat. Biol. 3 1 , 3 8 5 - - 3 8 9 Billen, D. and Hellermann, G.R. (1977) Chem. Biol. Interactions 18, 365--380 Thiehnann, H.W., Vosberg, H.P. and Reygers, U. (1975) EULr.J. Biochem. 5 6 , 4 3 3 - - 4 4 7 Thielmann, H.W. (1976) Ettr. J. Biochem. 6 1 , 5 0 1 - - 5 1 3 Masker, W.E. and Hanawalt, P.C. (1973) Nature New Biol. 244, 242--243 Masker, W.E. and Hanawalt, P.C. (1974) J. Mol. Biol. 88, 13--23 Masker, W.E. (1976) Biochim. Biophys. Acta 442, 162--173 Waldstein, E.A., Sharon, R. and Ben-Ishai, R. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2 6 5 1 - - 2 6 5 4 Dorson, J.W. and Moses, R.E. (1978) J. Biol. Chem. 2 5 3 , 6 6 5 - - 6 7 0 Billen, D. and Hellen~nann, G.R. (1974) Biochim. Biophys. Acta 3 6 1 , 1 6 6 - - 1 7 5 Clark, A.J. (1973) Ann. Rev. Genet. 7, 67--86 Moses. R.E. and Richardson, C.C. (1970) P~oc. Natl. Acad. Sci. U.S.A. 6 7 , 6 7 4 - - 6 8 1 Landbeck, L. and Hagen, U. (1973) Biochim. Biophys. Acta 3 3 1 , 3 1 8 - - 3 2 7 B~hner, R. and Hagen, U. (1977) Biochim. Biophys. Acta 479, 300--310 Weinstock, G.M., McEntee, K. and Lehman. I.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 7 6 , 1 2 6 - - 1 3 0 Waldstein, E.A. (1979) J. BacterioL 1 3 9 . 1 - - 7 Waldstein, E.A. and Setlow, R.B. (1976) Biochim. Biophys. Acta 442, 154--161