282 Nucleic Acids Research, 2005, Vol. 33, No. 1 alkali-labile sites (i.e. abasic sites) are detectable. The pres- ence of breaks allows supercoiled loops of DNA to relax and migrate to form a tail, and the fraction of DNA in the tail reﬂects the frequency of breaks. The number of SSB measured reﬂects the balance between breaks arising from BER and breaks resealed by the same repair process. As shown in Figure 1B, Pol b-defective cells treated in G1 phase, presented a higher level of SSB as compared with wild- type cells immediately after MMS treatment. Since MMS is a direct-acting mutagen, this is unlikely to reﬂect differential amounts of induced DNA damage but rather the fast formation and accumulation of SSB in G1 cells in the absence of Pol b. After a 1 h repair in drug-free medium (Figure 1C), the major- ity of breaks were repaired in wild-type cells, whereas mutant cells showed an accumulation of SSB. In contrast, the distribution of MMS-treated S phase cells according to the TM was very similar in normal and Pol b / cells (Figure 2), indicating that Pol b does not play a major role A B C A B C Figure 1. Effect of the lack of Pol b on MMS-induced DNA SSB repair during G1 phase. Comet assay was performed in wild-type and Pol b / cells, synchronized in G1 phase, after 15 min treatment with 0.5 mM MMS followed by 1 h repair in complete medium. The TM of 50 comets per experimental point was measured by computerized image analysis and the comets were classified in different classes (TM1-4) according to TM values. (A) Heterogeneity in DNA repair in G1 phase untreated cells, (B) in G1 phase MMS treated cells and (C) in G1 phase cells after repair. Figure 2. Effect of the lack of Pol b on MMS-induced DNA SSB repair dur- ing S phase. Comet assay was performed in wild-type and Pol b / cells, synchronized in S phase, after 15 min treatment with 0.5 mM MMS followed by 1 h repair in complete medium. The TM of 50 comets per experimental point was measured by computerized image analysis and the comets were classified in different classes (TM1–4) according to TM values. (A) Heterogeneity in DNA repair in S phase untreated cells (B) in S phase MMS treated cells and (C) in S phase cells after repair.