for Pol b are required (1–2 mM MMS) (Figure 4D). When the cells treated in this dose range were examined by pulse- ﬁeld gel electrophoresis (PFGE) DSB formation was con- ﬁrmed, but owing to the lower sensitivity of this technique, DSB were only detected in Pol b cells (data not shown). These results strongly suggest that, in the absence of Pol b, the accumulation of SSB in the G1 phase increases the like- lihood of DSB formation during replication fork progression. DSB, so generated, are a signal for the phosphorylation and consequently the formation of the g H2AX foci. This phenom- enon occurs also in wild-type cells and might explain the recombinogenic potential of MMS. Rad51 foci are induced in MMS-treated Pol b-defective cells The formation of g H2AX foci in Pol b defective cells prompted us to analyse the Rad51 protein. It is well known that Rad51 protein, which is essential for HR, relocalizes within the nucleus, in response to a variety of DNA damaging treatments, to form distinct foci that represent sites where repair reactions take place (29,30). Rad51 focus positive cells were observed in proliferating populations of Pol b / cells at early times after MMS treat- ment (2 h) (Figure 5C). The increase in focus positive cells observed in wild-type proliferating cells was less pronounced and appeared later in time (Figure 5C). In both cell lines, Nucleic Acids Research, 2005, Vol. 33, No. 1 285 the percentage of Rad51 positive cells reached a maximum at 8 h post-treatment and then declined to background values within 24 h. The presence of Rad51 foci in wild-type cells indicates that after MMS treatment recombination occurs. The elevated or accelerated assembly of Rad51 foci in Pol b / cells is consistent with the involvement of unrepaired SSB in recombination events by DSB formation. DISCUSSION SSB repair in Pol b mutant cells The clearest defects detected so far in Pol b mutant cells are the hypersensitivity to MMS and the reduced rate of MMS- induced SSB repair (3–6). Another mutant cell system shares these same features: the XRCC1 mutant rodent cells. XRCC1 protein plays a major role in SSB repair by interacting with multiple SSB repair partners, including PARP-1, Pol b, PCNA, DNA ligase III, APE-1 and polynucleotide kinase [reviewed in (31)]. Pol b is also known to interact with several proteins, largely overlapped to XRCC1 partners (32,33) and with XRCC1 itself (34,35). Biochemical and molecular mod- elling experiments suggest that the XRCC1–Pol b complex surrounds the SSB and helps recruiting the correct repair path- way (36). The striking similarity between the spectrum of Figure 5. Immunofluorescence analysis of Rad51 foci in Pol b proficient and deficient cells after MMS treatment. Wild-type and Pol b / cells were treated with 0.5 mM MMS for 15 min and then allowed to repair for the indicated periods of time. The spatial distribution of Rad51 foci is shown in representative nuclei of wild-type (A) and Pol b / (B) cells under high magnification. The histogram (C) reports the percentage of Rad51 foci. Cells with at least 10 foci were counted as Rad51 positive cells.