284 Nucleic Acids Research, 2005, Vol. 33, No. 1 which is the biological effect of the accumulation of these repair intermediates when replication occurs. We then exam- ined the effects of MMS treatment on cell cycle of Pol b- proﬁcient and deﬁcient cells. Proliferating cells were treated for 1 h with 1 mM MMS and then washed to remove MMS from the culture medium. In agreement with the previous studies (6,8), at 12 h post-treatment in Pol b / cells, the proportion of S phase increased signiﬁcantly (37% increase), whereas a mild effect was observed on the cell distribution of wild-type cells (18% increase of S-phase cells) (data not shown). The accumulation of unrepaired SSB in the absence of Pol b might determine a delay in the progression through the cell cycle. Next, g H2AX focus formation was investigated in prolif- erating MMS-treated cells. Histone H2AX is rapidly phos- phorylated in the chromatin microenvironment surrounding a DNA DSB, where it recruits repair and checkpoint protein complexes (28). As shown in Figure 4, the kinetics of g H2AX foci formation showed rapid DSB formation in Pol b-defective cells immediately after MMS treatment (0.5 mM for 15 min), whereas very few foci were detectable in wild-type cells. These foci gradually declined during repair time (Figure 4B). Moreover, the double staining with g H2AX and PCNA revealed that the g H2AX foci were at most localized in the nuclei of S phase cells (data not shown). In order to detect g H2AX foci in wild-type cells, doses higher than those used Figure 4. Immunofluorescence analysis of gH2AX foci in Pol b proficient and deficient cells after MMS treatment. Wild-type and Pol b / cells were treated with MMS and gH2AX foci analysis was performed. The spatial distribution of gH2AX 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 gH2AX foci after cell treatment with 0.5 mM MMS for 15 min and different repair times as indicated. The histogram (D) reports the percentage of gH2AX foci after cell treatment with increasing MMS doses as indicated. Cells with at least 10 foci were counted as gH2AX positive cells.