DNA polymerase β is required for efficient DNA ... - BioMedSearch

3040–3046 Nucleic Acids Research, 2000, Vol. 28, No. 16

© 2000 Oxford University Press

DNA polymerase β is required for efficient DNA strand break repair induced by methyl methanesulfonate but not by hydrogen peroxide Paola Fortini, Barbara Pascucci, Federico Belisario and Eugenia Dogliotti* Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Received May 25,2000; Revised and Accepted June 27, 2000

ABSTRACT The most frequent DNA lesions in mammalian genomes are removed by the base excision repair (BER) via multiple pathways that involve the replacement of one or more nucleotides at the lesion site. The biological consequences of a BER defect are at present largely unknown. We report here that mouse cells defective in the main BER DNA polymerase β (Pol β) display a decreased rate of DNA single-strand breaks (ssb) rejoining after methyl methanesulfonate damage when compared with wild-type cells. In contrast, Pol β seems to be dispensable for hydrogen peroxide-induced DNA ssb repair, which is equally efficient in normal and defective cells. By using an in vitro repair assay on single abasic site-containing circular duplex molecules, we show that the longpatch BER is the predominant repair route in Pol βnull cell extract. Our results strongly suggest that the Pol β-mediated single nucleotide BER is the favorite pathway for repair of N-methylpurines while oxidationinduced ssb, likely arising from oxidized abasic sites, are the substrate for long-patch BER. INTRODUCTION DNA apurinic/apyrimidinic (AP) sites are ubiquitous lesions that arise either from spontaneous depurination or by removal of damaged bases produced by physical and chemical treatment. Because of their ‘non instructional’ character these lesions are likely to be both cytotoxic and mutagenic. Prokaryotes and eukaryotes have evolved a defense mechanism to repair this kind of DNA damage and to guarantee the integrity of the genome, known as base excision repair (BER). For many years it has been believed that the biochemistry of the BER was completely understood. Recent data have triggered again the interest in this excision repair process by showing the existence of alternative BER pathways which involve the replacement of one (short patch BER) or more nucleotides (long patch BER) at the lesion site (for a review see 1). Both BER pathways have been reconstituted in vitro by using purified mammalian proteins (2–5).

The DNA polymerase of election for the short patch BER is DNA polymerase β (Pol β). This enzyme is able to release the remnant 5′ deoxyribosephosphate formed by the major mammalian AP endonuclease HAP1 and to fill in the gap (for a review see 6). Conversely, in the long-patch BER either Pol β (7,8) or Pol δ/ε (9,10) are able to perform the resynthesis step. Besides Pol δ/ε, other replication factors are specifically involved in this pathway: the structure-specific endonuclease FEN1, the proliferating cell nuclear antigen (PCNA) and the replication factor C responsible for the loading of PCNA onto the DNA template. Notwithstanding the progress made in the biochemical characterization of BER, the biological significance of this repair process is largely unknown. Since no human BER mutants are available, our knowledge relies upon mice lacking a specific DNA repair activity. Whereas HAP1 endonuclease, Pol β, DNA ligase I and XRCCI gene disruptions lead to lethality during embryogenesis, the DNA glycosylase-deficient embryos (uracil DNA glycosylase, 3-methyladenine DNA glycosylase and 7,8-dihydro-8-oxoguanine-DNA glycosylase) are viable and no obvious phenotype is observed (for a review see 11). Null cells can be derived from embryos of mice carrying lethal mutations. Embryonic fibroblast cell lines homozygous for a deletion mutation in the Pol β gene have been established (12) from Pol β-knock out mice. In this paper we show that a defect in Pol β leads to hypersensitivity to the cytotoxic effects of both methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2) and to the accumulation of MMS-induced DNA single-strand breaks (ssb). In contrast, Pol β is not required for DNA ssb repair induced by H2O2. Biochemical studies clarify that the long patch BER is the predominant AP site repair route in the Pol β-null extracts. These data strongly suggest that long-patch BER is the predominant repair route for ssb arising from oxidative damage. MATERIALS AND METHODS Chemicals MMS (Merck, White House Station, NJ) was dissolved in dimethyl sulfoxide (DMSO) shortly before use and quickly diluted in serum-free medium to the required concentrations (final concentration of DMSO 1 nt. The long-patch BER extended beyond the T2 as shown by the presence of radioactivity in the B and C fragments (lanes 3–6) although a progressive decrease in the number of substituted nucleotides was observed at increasing distances from the lesion site. Quantitation of the radioactivity in the different restriction fragments (Fig. 4B) showed that in wild-type cell extracts 1 nt replacement events represented almost 70% of all repair reactions with the remaining 30% involving resynthesis of longer patches. Conversely, mapping of the repair patches in Pol β-null extracts showed that the level of dCMP incorporation in the A fragment (Fig. 4A, lane 7) was slightly higher (20%) than that of dTMP in the same fragment (Fig. 4A, lane 8) (Fig. 4B). Similar levels of radioactivity were detected over T2, C3 and T4 (Fig. 4B). This implies that, in the absence of Pol β, the single nucleotide replacement reactions are unfavored and the majority of the repair patches extend at least over the first 4 nt including the original lesion site. Moreover, a significant level of incorporation was detected in the C fragment (Fig. 4A, lanes 11 and 12) testifying that a significant fraction of the displaced and then resynthesized fragments are >9 nt in length. The type of DNA polymerase involved in the BER reaction is therefore preferentially associated with a specific BER branch: Pol β with the single nucleotide BER and Pols other than Pol β, likely Pol δ/ε, with the long-patch BER. When the distribution of the long (≥2 nt) repair patches in Pol β-proficient and -deficient cells was compared (Fig. 4C) a predominance of 2 nt replacement events was observed in normal cells while defective extracts were characterized by a relatively higher fraction of patches extending above T9. The long-patch repair kinetics are similar in Pol βproficient and -deficient cell extracts In order to better characterize the BER process performed by normal and defective Pol β cells, the rate of nucleotide resynthesis was investigated at the lesion site and along the sequence

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Figure 4. Mapping at nucleotide resolution of the repair patches at a single abasic site by wild-type and Pol β-null cell extracts. (A) Autoradiograph of a denaturing polyacrylamide gel. Whole cell extracts from Pol β-proficient and -deficient cells were used in an in vitro repair assay on circular duplex DNA molecules containing a single abasic site. Repair replication was performed for 1 h in the presence of [α-32P]dCTP or [α-32P]dTTP at 30°C. DNAs were digested with: SacI–XbaI (fragment A) to release the 17 bp fragment originally containing the lesion, XbaI–PstI (fragment B) and SalI–PstI (fragment C) to release the 16 bp and 10 bp fragments respectively, containing nucleotide residues 3′ to the AP site. IS, internal standard. (B) The levels of incorporation in the restriction fragments were measured by electronic autoradiography and corrected for DNA recovery (net c.p.m.). The incorporation at C3 and T4 was calculated by subtracting the radioactivity (dCMP or dTMP incorporation) of fragment C from that of fragment B. (C) Distribution of the radioactivity present in the long (≥2 nt) repair patches along the sequence 3′ to the AP site. Top, scheme of the restriction mapping. X, position where the AP site was originally located.

3′ to it. As shown in Figure 5, the single nucleotide replacement events, which are the hallmark of wild-type whole cell extracts, are fast processes that reached a plateau in 20 min (Fig. 5A, lanes 1–3; Fig. 5C). These data confirm previous reports on the higher efficiency and velocity of these reactions as compared to the long-patch BER in wild-type cell extracts (9).

Figure 5. Mapping at nucleotide resolution of the repair patches at a single abasic site as a function of time by wild-type and Pol β-null cell extracts. (A and B) Autoradiograph of a denaturing polyacrylamide gel. Whole cell extracts from Pol β-proficient (A) and -deficient (B) cells were used in an in vitro repair assay on circular duplex DNA molecules containing a single abasic site. Repair replication was performed for the indicated periods of time (10–60 min) in the presence of [α-32P]dCTP or [α-32P)]dTTP at 30°C. DNAs were digested with: SacI–XbaI (fragment A) to release the 17 bp fragment originally containing the lesion and XbaI–PstI (fragment B) to release the 16 bp containing nucleotide residues 3′ to the AP site. IS, internal standard. (C) The levels of incorporation in the restriction fragments were measured by electronic autoradiography and corrected for DNA recovery (net c.p.m.). Top, scheme of the restriction mapping. X, position where the AP site was originally located.

What is the efficiency of the long-patch BER in Pol β defective cells? Pol β-null extracts, which perform predominantly long-patch BER, present the same long-patch repair time course as that detected in normal extracts (Fig. 5A and B, lanes 4–12). After 10 min repair, while the one gap-filling reactions were almost at plateau in wild-type extracts (Fig. 5A, lane 1), the repair reactions of multiple nucleotide substitutions were barely detectable on the autoradiography (Fig. 5A, lanes 4, 7 and 10; Fig. 5B, lanes 1, 4, 7 and 10). In both extracts there was

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a time-dependent linear increase of the total yield of long patch repair products (Fig. 5C) with a 4-fold increase over 1 h repair. The slow and comparable time courses of the long-patch BER in both cell extracts suggest that the repair machinery involved, if different, presents similar rate limiting steps, either the strand displacement reaction per se or the assembly of a multi-protein complex at the lesion site. DISCUSSION Pol β-dependent cytotoxicity induced by MMS and H2O2 Mouse cells defective in Pol β are hypersensitive to a variety of monofunctional alkylating agents (12,16). In this study we confirm the hypersensitivity to the cytotoxic effects of MMS and we present evidence of cross-sensitivity to H2O2. This oxidative agent causes damage to nuclear DNA by generating hydroxyl radicals in close proximity to DNA. The reaction of these reactive oxygen species (ROS) with DNA results primarily in base modifications but also in base loss and ssb due to fragmentation of the sugar. The increase in the cytotoxic effect of H2O2 associated with the loss of Pol β is lower as compared with that observed after methylation damage (compare Fig. 1B and A). This is compatible with (i) a minor role of Pol β in the processing of ROS-induced lesions; and/or (ii) with the production of repair intermediates with a lower cytototoxic potential as compared with those created during the processing of methylpurines. We (17) and others (18–20) have previously shown that Pol β is the favorite Pol for one gap filling reactions following removal by bifunctional DNA glycosylase/AP lyases of the main oxidized purines (i. e. 8-oxoG) and pyrimidines (i.e. thymine glycol) induced by ROS (for a review see 21). Pol δ/ε can eventually repair these gaps but at a slower rate (17). The absence of Pol β might then cause the persistence of cytotoxic strand interruptions arising from an inefficient repair of oxidized DNA bases. These strand breaks, which present a genuine 5′ nucleotide, might exert a lower lethal effect as compared with ssb with a 5′ blocked terminus as those produced during BER of methyl-purines. Pol β-dependent DNA ssb repair induced by MMS We report here that mammalian DNA ssb repair induced by MMS is affected by the deletion in the Pol β gene. The accumulation of DNA ssb observed after MMS treatment is likely to be responsible for the increased cytotoxicity observed in Pol β-null cells after methylation damage. A similar phenotype, i.e. increased MMS-sensitivity killing (22) and accumulation of alkylation-induced ssb (23), has been reported for XRCC1defective cells. Interestingly, the defect in DNA ssb repair was already remarkable after 30 min incubation with MMS indicating that Pol β is required for efficient ssb repair at early times after base damage. After 2 h repair, although a higher level of ssb is still detectable in the Pol β mutant cells, the majority of breaks (70%) are resealed in these cells via a Pol βindependent process. This is in contrast with what is observed when XRCC1 is defective: the amount of ssb detected after 3 h repair following ethyl methanesulfonate (EMS) damage is even higher than the level detected immediately after DNA damage presumably reflecting continued base excision or exonuclease activity (24) in the absence of the ligation step. These data provide strong evidence that in vivo MMS damage

is mainly processed via a Pol β-dependent pathway but, if Pol β is not functional, a Pol δ/ε-dependent back-up system can replace the main pathway limiting the hazardous effect of unrepaired ssb. Pol β-independent DNA ssb repair induced by H2O2 We report here that DNA ssb repair induced by ROS is unaffected by Pol β mutation. What is the origin of H2O2-induced ssb detected by alkaline SCGE? In addition to frank strand breaks, which are a minor fraction of the total breaks, the majority of induced ssb present 5′ and 3′ phosphate termini implying that the release of a base has occurred (25). These abasic sites are 10% regular abasic sites and the majority are oxidized abasic sites, mainly 4′ sites (26). The contribution to the total yield of detected ssb of abasic sites arising from the processing of base damage like thymine glycol or 8-oxoG, should be limited due to the slow repair kinetics of these lesions (27). We (17) and others (18,19) have previously shown that the selection of the BER pathway is strongly affected by the structure of the termini of the abasic sites and in particular the occurrence of long-patch BER is favored by the presence of reduced or oxidized AP sites (3). In vitro the longpatch BER is performed equally well either by Pol β (3,8) or Pol δ/ε (10,4) . This might explain why ROS-induced DNA ssb repair is independent of a functional Pol β gene. BER pathways in Pol β-null cell extracts The fine mapping of the repair patches occurring at abasic sites when Pol β-defective cell extracts are used in the in vitro BER assay provided the first evidence that the predominant route of repair in the absence of Pol β is the long-patch BER. Since Pol δ/ε are able to perform the 1 nt polymerization step at sites of base loss (4,10) the irreplaceable function of Pol β, which is required for single nucleotide BER, should be the dRPase activity. The absence of this function and therefore the persistence of the 5′ blocked terminus is likely to be responsible for the adverse biological effects (cytotoxicity) detected in Pol β null cells treated with MMS. On the other hand, the modest (as in the case of MMS) or absent (as in the case of H2O2) effect of a defective Pol β on DNA ssb repair supports the conclusion that Pol δ/ε is capable of BER in vivo. Interestingly, studies performed more than one decade ago in permeable cell systems (reviewed in 28) by using DNA Pol inhibitors showed that either aphidicolin (Pol δ/ε inhibitor) or ddTTP (Pol β inhibitor) were able to partially inhibit alkylation damageinduced repair (29–31) while ionizing radiation-induced DNA damage was specifically inhibited by aphidicolin (32). Our data are in full agreement with these observations. The repair of damage by monofunctional alkylating agents involves preferentially Pol β but occurs, although less efficiently, also in its absence, while ROS-induced DNA ssb are efficiently repaired via Pol δ/ε-mediated long-patch BER. The comparison of the long repair patch profile in wild-type versus Pol β null cell extracts showed that, although there was a significant overlapping in patch size distribution between the two extracts, the ‘short’ patches (mainly 2 nt long) were more represented in wild-type extracts while ‘long’ patches (up to 15 nt) were more frequent in Pol β-null extracts. The repair patch size distribution observed is in agreement with the different ability to perform processive gap-filling synthesis of Pol β (up to 6 nt ) (33,34) and Pol δ/ε (10–18 nt) (4). Pol β has

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been implicated in long-patch BER (7,35) and the excision of a characteristic dRP-trinucleotide was reported. This in contrast with the repair patch profile that we observed. Differences between the DNA substrates used in these studies, like the lesion sequence context, might explain this discrepancy. The time course of the repair process was similar in the two cell extracts suggesting that, if the repair machinery is different, there is a common rate-limiting step (the formation of the flap structure?). These findings are compatible with a model where both Pols are able to participate to the resynthesis step in the BER process. If DNA pols are functionally interchangeable in the repair synthesis step the challenging question is what are factors that control their participation to the BER process. Recent evidence has been provided that specific BER complexes might be acting at different stages of the cell cycle (24,36). It is tempting to speculate that the overlapping between the factors involved in DNA replication and those required for long-patch BER might favor Pol δ/ε in BER at post-replicative stages while the single nucleotide Pol β-mediated BER might be favored in pre-replicative stages. Future research should investigate whether the involvement of the two Pols in BER is indeed controlled by the cell cycle stage. ACKNOWLEDGEMENTS We are grateful to M. Stucki for the biochemical characterization of the cell extracts and to U. Hubscher for helpful discussions. This work has been partially supported by the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). REFERENCES 1. Lindahl,T. and Wood,R.D. (1999) Science, 286, 1897–1905. 2. Kubota,Y., Nash,R., Klungland,A., Schar,P., Barnes,D. and Lindahl,T. (1996) EMBO J., 15, 6662–6670. 3. Klungland,A. and Lindahl,T. (1997) EMBO J., 16, 3341–3348. 4. Pascucci,B., Stucki,M., Jonsson,Z.O., Dogliotti,E. and Hubscher,U. (1999) J. Biol. Chem., 274, 33696–33702. 5. Matsumoto,Y., Kim,K., Hurwitz,J., Gary,R., Levin,D.S., Tomkinson,A.E. and Park,M.S. (1999) J. Biol. Chem., 274, 33703–33708. 6. Wilson,S.H. (1998) Mutat. Res., 407, 203–215. 7. Dianov,G., Prasad,R., Wilson,S. and Bohr,V. (1999) J. Biol. Chem., 274, 13741–13743. 8. Prasad,R., Dianov,G.L., Bohr,V.A. and Wilson,S.H. (2000) J. Biol. Chem., 275, 4460–4466.

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