To dissect the mechanism and the consequences of SSB accumulation in the absence of Pol b, we investigated the processing of these intermediates during different phases of the cell cycle. We ﬁnd that, after MMS treatment, the accu- mulation of SSB in Pol b / cells occurs speciﬁcally during the G1 phase of the cell cycle. These unrepaired SSB trigger the formation of chromatin-bound PCNA complex in the cell nuclei. When replicating, Pol b-defective cells are treated with MMS, gH2AX foci are rapidly formed, indicating that the conversion of BER intermediates into DSB occurred during the S phase. Subsequently, Rad51 relocalizes within the Pol b / cells nuclei to form foci. These ﬁndings indicate that, in the absence of Pol b, the unrepaired SSB produced during BER can act as substrates for the HR pathway to avoid excess- ive cytotoxicity. MATERIALS AND METHODS Cell culture, cell cycle synchronization and cell cycle analysis SV40 transformed wild-type and Pol b-deﬁcient mouse embryonic ﬁbroblasts (a gift from Dr S. H. Wilson; NIEHS, Research Triangle Park, NC) (3) were cultured in DMEM with glutamax-1 supplemented with different concentrations of foetal calf serum (FCS; see below), penicillin (100 U/ml), streptomycin (100 mg/ml) and hygromycin (80 mg/ml) at 34 C in a 10% CO2 incubator. Wild-type and Pol b / cells were synchronized in G1 and S phases by growth in a low serum (0.5%) containing medium for 24 h. They were then incubated in complete medium (10% FCS) in addition to dif- ferent concentrations of mimosine. In particular, G1 phase cells were obtained by incubation with 200 mM mimosine for 16 h and S phase cells by incubation with 100 mM mimosine for 16 h followed by 6 h recovery in the complete medium. Cells were then trypsinized, ﬁxed, stained with pro- pidium iodide and analysed in an EPICS XL cytoﬂuorimeter (Coulter). The proportion of cells in G1 and S phase was quantiﬁed using the MCYCLE software (Coulter). Single cell gel electrophoresis (SCGE) G1 and S phase synchronized cell cultures were treated with 0.5 mM MMS in the serum-free medium for 15 min. Cells were allowed to repair DNA damage by incubation in com- plete medium for 1 h. DNA breaks were detected as described previously (20). Brieﬂy, cells were embedded in agarose gel and then spread on a frosted microscope slide. Cells were lysed in 2.5 M NaCl, 10 mM Tris–HCl, 100 mM Na2EDTA, 1% Triton and 10% dimethyl sulfoxide, pH 10 for 1 h at 4 C. After lysis, the cells were pre-incubated for 20 min at 4 C in the electrophoresis buffer (0.3 M NaOH and 1 mM Na2EDTA, pH 13.5) and then subjected to alkaline gel electrophoresis (in 300 mA at 4 C for 20 min). After SCGE, cell DNA was stained with ethidium bromide and visualized using ﬂuores- cence microscopy. Slides were analysed by a computerized image analysis to quantitate the DNA damage. The tail moment (TM), calculated by multiplying the total intensity of the comet tail by the migration distance from the centre of the comet head, was used as a measure of the DNA damage. The comets were classiﬁed into different classes (from 1 to 4) Nucleic Acids Research, 2005, Vol. 33, No. 1 281 corresponding to increasing TM values (21). A total of 50 cells for each experimental point were scored blind from two slides. Immunofluorescence microscopy Wild-type and Pol b / cells in G1 phase were treated with 0.5 mM MMS for 15 min and then allowed to repair for the indicated periods of time. For the detection of chromatin- bound PCNA complex formation, the cells were rinsed twice in phosphate-buffered saline (PBS), ﬁxed in ice-cold methanol for 20 min at 20 C, and then incubated for 30 s with ice-cold acetone to extract the soluble PCNA fraction. Cells were subsequently washed twice with PBS and incubated for 1 h at room temperature with primary anti-PCNA antibody (PC10 from Santa Cruz; 1:50). Subsequently, the cells were washed with PBS and incubated for 45 min with TRIC- conjugated antimouse IgG. After washing three times for 5 min with PBS, cells were mounted with glycerol. No sig- niﬁcant signal attributable to secondary antibody alone was detected. The same experimental procedure was used to detect gH2AX foci. Cells were incubated with the primary anti- gH2AX antibody (gH2AX; Trevigen, Gaithersburg, MD) in the ratio 1:100 overnight at 4 C in a humidiﬁed incubator, washed with PBS and incubated for 45 min with ﬂuorescein isothiocyanate (FITC)-conjugated antirabbit IgG at 37 C. For the detection of chromatin-bound PCNA complex and gH2AX positive cells, the dual labelling was performed using the same experimental procedure, which is used for the single labelling of PCNA complex and gH2AX foci. For the detection of Rad51 foci, an aliquot of the cell suspension was centrifuged onto the clean glass slides at 600 r.p.m. for 10 min, in a cytospin. After cytocentrifugation, the slides were ﬁxed in 20 C methanol for 20 min and then immersed in ice-cold acetone for a few seconds to permeabilize the cells for anti- body staining. Following three washes with PBS, the prepara- tions were blocked with 10% normal goat serum (Hyclone Laboratories) in PBS for 1 h, incubated at 4 C overnight with the primary anti-Rad51 antibody (Rad51, Ab1, polyclo- nal, Oncogene; 1:200), washed in PBS and then incubated for 45 min with FITC-conjugated antirabbit IgG at 37 C. All the preparations were counterstained with 0.1 mg/ml of 40,6-diamidino-2-phenylindole (DAPI). Coverslips were analysed using a Leica DRMB ﬂuorescence microscope equipped with a charge coupled device camera. The images were processed using the IAS 2000 Delta System software (Adobe, San Jose`, CA). RESULTS MMS-induced SSB repair is impaired in G1-treated Pol b-defective cells Mouse ﬁbroblasts lacking Pol b present a reduced rate of MMS-induced SSB repair (3–6). To elucidate this mechanism better, MMS-induced SSB repair was analysed in G1 and S phase synchronized wild-type and Pol b / cells. A signiﬁcant enrichment of G1 and S phase cells ( 60–70% of the total cell population) was obtained in both cell lines by serum starvation and mimosine treatment (data not shown). Cells were then treated with MMS (0.5 mM) for 15 min and allowed to repair for 1 h in fresh medium. The level of SSB was measured by alkaline SCGE. Under these conditions, both DNA SSB and