in the fast repair reactions occurring in S phase cells. It is of note that MMS treatment (Figure 2B) of the S phase Pol b cells resulted in a mild shift of the TMs to the right, in comparison with wild-type cells, indicating that DNA was fragmented to a slightly higher and more varied degree. At 1 h post-treatment (Figure 2C), both cell lines showed a persistence of DNA breaks but the yield of unrepaired breaks was similar in the two cell lines. The comet assay allows the detection of both SSB and DSB. Therefore, it is possible that the unrepaired breaks observed after the 1 h repair of S-phase-treated cells are DSB formed by the progression of the replicative fork in the presence of MMS-induced lesions and/or repair intermediates. These ﬁndings assign the defect in SSB rejoining of Pol b cells to DNA repair that occurs during the G1 phase, strengthening the notion that Pol b operates during a pre- replicative BER. Chromatin-bound PCNA complex formation is triggered by MMS damage in Pol b cells It is known that the chromatin-bound PCNA complex forma- tion is triggered by DNA damage in quiescent cells in G1 and G2 phases. This occurs following treatment with various DNA damaging agents like UV light (22–24), X-rays (25), alkylat- ing agents (26) and hydrogen peroxide (26,27). We asked whether the accumulation of SSB in G1 Pol b cells Nucleic Acids Research, 2005, Vol. 33, No. 1 283 could also be revealed by PCNA complex formation. Pol b- proﬁcient and deﬁcient cell lines synchronized in G1 phase were treated with MMS, and PCNA foci were analysed by immunoﬂuorescence. In untreated cells, the chromatin-bound PCNA was barely detectable (Figure 3A and B). In Pol b / cells, the PCNA complex appeared to form rapidly during treatment, since the cells ﬁxed immediately after 15 min incubation with MMS (no recovery time) showed an intense chromatin-bound PCNA foci staining (Figure 3B and C). In contrast, wild-type cells did not show any PCNA staining immediately after treatment (Figure 3C). The higher level of SSB in G1 Pol b / cells is therefore associated with chro- matin-bound PCNA complex formation. The analysis of PCNA immunostaining at different post-treatment incubation times showed that PCNA foci also appeared in wild-type cells but at later times as compared with Pol b / cells. In both cell lines, the percentage of PCNA foci reached a peak at 15 min after MMS treatment and then gradually declined (Figure 3C). These data indicate that, following MMS damage, PCNA foci accumulate in Pol b-defective cells, consistent with the accumulation of SSB in G1 phase cells. gH2AX foci are induced in MMS-treated Pol b-defective cells Since G1-treated Pol b / cells present a higher level of SSB when compared with wild-type cells, we raised the question Figure 3. Immunofluorescence analysis of chromatin-bound PCNA in Pol b proficient and deficient cells after MMS treatment. Wild-type and Pol b / cells, synchronized at G1 phase, were treated with 0.5 mM MMS for 15 min and then allowed to repair for the indicated periods of time. Cells were fixed in cold methanol and then in cold acetone to extract the soluble PCNA fraction. The spatial distribution of PCNA 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 PCNA foci. Cells with at least 5 foci were counted as PCNA positive cells.