286 Nucleic Acids Research, 2005, Vol. 33, No. 1 Table 1. Genetic instability of XRCC1 and Pol b defective cell lines Phenotype Sensitivity to MMS Sensitivity to H2O2 Sensitivity to IR SSB repair DSB repair SCE CA Mutation frequency (MF) aData from (31). bData from (4,53,54). XRCC1 mutanta Highly increased Increased Slightly increased Reduced rate after IR, EMS, MMS Reduced rate after X-rays Increased spontaneous and induced SCE Increased spontaneous and induced CA Increased spontaneous and induced MF Pol b mutantb Increased No difference No difference Reduced rate after MMS Reduced rate after MMS Increased induced SCE Increased induced CA Increased induced MF altered functions in Pol b and XRCC1 mutant cells (Table 1) is therefore not surprising. It should also be mentioned that cells defective in the DNA nick sensor protein, PARP-1, share several features with Pol b/XRCC1 mutants (reviewed in 37). Interestingly, recent data suggest that PARP-1 plays a role as a surveillance protein for a stalled BER intermediate and an important regulator factor in BER sub-pathway choice (38,39). The extensive work of characterization of XRCC1- dependent SSB repair as a function of cell cycle by Caldecott and co-workers (40) has allowed the identiﬁcation of different SSB repair pathways that operate in different phases of the cell cycle: a rapid SSB repair that appears to operate throughout interphase (likely overlapped with the so-called short patch BER) and an S/G2 speciﬁc SSB repair. It has been speculated that this second pathway, which is coordinated with DNA replication, employs long-patch BER proteins like PCNA, Fen 1 and DNA ligase I (41). In this study, we show that Pol b has a speciﬁc role in the rapid SSB repair that occurs in G1 phase and also requires an XRCC1–Lig3a interaction (40). In contrast, we show that S phase-treated cells repair SSB efﬁciently, independent of Pol b. On the basis of these data, we hypothesize that the second XRCC1-dependent pathway, which operates speciﬁcally in S/G2, does not involve Pol b but replicative DNA polymerases. This model gives a possible explanation for the more severe phenotype of XRCC1 defect- ive cells as compared with Pol b mutant cells (see Table 1): XRCC1 is an essential component of SSB repair in all phases of the cell cycle, whereas DNA polymerases other than Pol b operate during replication-associated SSB repair. It is there- fore the defect in the S/G2 speciﬁc XRCC1-dependent (Pol b independent) SSB repair that accounts for the higher genetic instability of XRCC1 defective cells as compared with Pol b defective cells after DNA damage. It is well known that the treatment of quiescent cells with UV light (22–24) as well as IR (25), triggers a rapid transition of PCNA from a soluble to an insoluble form, which can be visualized by immunoﬂuorescence. This transition correlates with nucleotide excision repair and BER capacity, respect- ively. In the case of BER, the PCNA complex formation has been reported to correlate with BER kinetics (42). Our data are in line with these previous ﬁndings. The PCNA distribution kinetics closely resemble that of SSB repair, showing a sig- niﬁcant increase in PCNA complex formation in Pol b cells after MMS damage and a slower decrease as a function of time as compared with wild-type cells. Interestingly, a previous study (43) showed no difference in the PCNA complex forma- tion in Pol b cells exposed to X-rays as compared with wild-type cells. Altogether, these data indicate that Pol b is the major Pol in pre-replicative SSB repair following alkylation damage but other pathways, which are Pol b independent, are involved in SSB repair after oxidative DNA damage (5). gH2AX and RAD51 foci formation in Pol b mutant cells If cells enter into the S phase with a load of unrepaired SSB, the likelihood of DSB formation is expected to increase. We show for the ﬁrst time that the repair defect of Pol b G1 cells is detectable by using gH2AX foci analysis. The majority of MMS-induced gH2AX foci were present in PCNA positive cells and thus these foci are attributed to replication-mediated DNA damage. Conversely, in the case of an agent able to induce direct DSB as IR, these foci are observed in both PCNA positive and negative cells (9). Several studies indicate that gH2AX foci per cell are proportional to the number of introduced DSB (44,45). A recent study (46) suggests that lesions other than DSB induced by monofunctional alkylating agents might lead to gH2AX foci formation. Pol b cells show a signiﬁcant fraction of gH2AX nuclear foci in PCNA positive cells immediately after MMS treatment (when wild- type cells show background levels). By using an alternative technique such as PFGE to measure DSB, we were able to conﬁrm the induction of DSB by MMS in Pol b cells, although MMS doses higher than those used for gH2AX foci analysis were required to detect an effect. We can thus con- clude that MMS-induced gH2AX foci in Pol b cells are likely to reﬂect replication-mediated DSB formation. More- over, we conﬁrm that gH2AX foci analysis is the most sens- itive assay for DSB formation (45). Almost 50% of gH2AX foci are still present 2 h after MMS treatment. It is well known that DSB repair presents a biphasic kinetics with a rapid and a slow phase (the bulk of the breaks are repaired within 1–2 h and the remaining breaks are repaired within 5 h) (47). The kinetics of gH2AX foci disappearance in our cells suggests that MMS-induced DSB are repaired by the ‘slow’ repair system that has been hypothesized to involve HR (48). Repair of DSB may be largely error-free by HR but they are also a potential substrate for NHEJ, which may introduce a large number of mutations (49). It is known that Rad51 is essential for HR and, in response to DNA damage, the protein reloca- lizes within the nucleus to form foci (29,30). These foci are thought to represent sites where repair takes place. Rad51 foci assemble at earlier times in Pol b defective cells when com- pared with wild-type cells. Since Rad51 foci that are formed after DNA damage mark a subset of cells that have entered the HR pathway, we could conclude that BER intermediates, accumulated in the genome as a consequence of a defective BER, could act as substrates for HR by DSB formation. The presence of gH2AX foci (at high MMS doses) and of Rad51 foci in MMS-treated wild-type cells deserves a comment. It is known that MMS is recombinogenic (29,50). The detection of gH2AX foci strongly suggests that MMS is recombinogenic via DSB formation triggered by unrepaired BER intermediates. This same phenomenon would be ampli- ﬁed in the absence of Pol b due to accumulation of unrepaired intermediates.