6616 Nucleic Acids Research, 2012, Vol. 40, No. 14 Figure 6. Reduced chromosomal SSBR in MCSZ LCLs. (A) The indicated LCLs were mock-irradiated or g-irradiated (20 Gy) and incubated for the indicated time periods at 37 C to allow DNA repair. Levels of chromosomal DNA strand breaks were quantiﬁed by alkaline comet assays as average tail moments. Data are the mean of 100 cells/time point per experiment and are the average of at least four independent experiments (±SEM). Data sets for the MCSZ LCLs MC22601, MC22602 and MC5403 were signiﬁcfantly different (two-way ANOVA) to those for the related WT LCLs MC22606 (P = 0.000691), MC22604 (P = 0.00158) and MC5405 (P = 0.00802), respectively. (B) The indicated LCLs were mock-treated or treated with 14 mM CPT for 60 min at 37 C and the level of accumulated chromosomal DNA strand breaks quantiﬁed by alkaline comet assays as average tail moments, as described above. Data are the mean of 100 cells/time point and are the average of at three independent experiments (±SEM). DNA sets for the MCSZ LCLs MC22601 and MC22602 were signiﬁcantly different (two-way ANOVA) to those for the related WT LCLs MC22606 (P = 0.00172) and MC22604 (P = 1.498 E-05), respectively. Note that the LCL line 1635 is from an individual with the Tdp1-mutated disease, SCAN1 and is included as a positive control. (C) The indicated LCLs were mock-treated or treated with 14, 28 or 56 mM CPT for 60 min at 37 C and analysed as above. Only the data set for the MCSZ LCL MC22602 was signiﬁcantly different from WT (MC22604; P = 3.221 E-06). WT. In contrast, these proteins exhibited little DNA 50- kinase activity, under the same conditions. The absence of signiﬁcant DNA kinase activity in PNKPT424Gfs48X and PNKPexon15 fs4X is not surprising, because of their extensive deletions within the DNA kinase domain. However, it is surprising that the speciﬁc activity of the DNA phosphatase domain is normal, under the condi- tions examined, because in WT PNKP the phosphatase