TI - Discussion . AB - Here we report on extensive strand resection or unwinding of DSB ends , resulting in long ssDNA ends , in NHEJ deficient cells >= 1 h after DSB induction . The cause of this processing of DSB ends is not known , but one possibility is an alternative DSB repair mechanism , which attempts to rejoin the DSBs in the absence of NHEJ . Since these ssDNA ends were only detected in the S phase but not in the G1 phase cell cycle , HR is an obvious candidate . HR is downregulated in the G1 phase but active in the S and G2 phases and the first step in HR is the creation of ssDNA ends , which may extend as far as 1 kbp from the break [31] . CDK1 probably has an important role in this regulation and in budding yeast CDK1 is required for DSB -induced HR and efficient resection of DSB ends [18] . Several reports suggest that NHEJ and HR compete for the rejoining of DSBs ; Ku proficient cells show a lower degree of HR than Ku deficient cells [32,33] , supposedly as a result of the protection of ends from HR processing by Ku . This is supported by the observation that the initiation of HR is blocked by the binding of Ku to DSB ends [34] . Upregulation of HR has also been demonstrated in XRCC4 deficient cells [17,35] , indicating an ability of HR to compensate for non-functional NHEJ . In addition , data from cells containing different mutations in DNA-PKcs autophosphorylaTION sites suggest that the PHOSphorylation status of DNA-PKcs may have a role in regulating the access of ends and might control the choice of pathway [36] . Interestingly , HR was blocked when the catalytic activity of DNA-PKcs was inhibited , presumably as an effect of the blocking of the end processing required for HR [37] . This is in agreement with the present finding that ssDNA formation at DSB ends was blocked in DNA-PKcs inhibited cells , although we showed that DNA Ligase IV/XRCC4 was also needed at the ends together with inhibited DNA-PKcs to prevent processing ( Fig 4 and 5 ) . However , whether it is HR , including single strand annealing ( SSA ) between repetitive DNA sequences , or another process that gains access to the DSB ends in the absence of functional NHEJ and is responsible for the generation of ssDNA ends , that process is clearly not able to completely compensate for the lack of NHEJ , as a large number of unrejoined DNA fragments were still present 4-24 h after DSB induction in NHEJ deficient cells . Thus NHEJ seems to be the major repair pathway in mammalian cells throughout the cell cycle . The ssDNA formation at unrejoined DSBs in NHEJ deficient cell lines has major implications for the quantification of DSBs by the common PFGE assay , where Mbp-DNA fragments are separated by electrophoresis . In the standard assay , naked DNA fragments are extracted from cells by incubating the cells in EDTA buffer containing a detergent and Proteinase K at 50degC . We show here that incubation at 50degC causes the ssDNA ends to fuse , resulting in fewer DNA fragments and an underestimation of the number of DSBs still present 1-24 h after DSB induction . The use of a recently developed assay [26] , in which the DNA extraction is performed at 0degC prevents heat -dependent ssDNA hybridization and therefore results in a more accurate DSB quantification and in some cases a drastic decrease in the estimated rejoining capability of NHEJ deficient cell lines (Fig 1B) . Although larger numbers of DSBs were detected with the cold protocol in all NHEJ deficient cell lines tested , the differences in the rejoining capacity observed between these cell lines with the standard protocol remained when the new cold protocol was used . The hamster cell lines V3 and Irs20 , although displaying a much slower repair of DSBs compared to wild type cells , still rejoined the majority of breaks within 24 h . The same ability for rejoining was detected in asynchronous and G1-synchronized V3 cells ( compare Figure 1B with Figure 6A ) , suggesting that a DNA-PKcs independent repair pathway , separated from HR and S-phase dependency , exists in the V3 cell line . In contrast to the hamster cells , no or very little rejoining of DSBs was detected up to 24 h of repair in the human glioma cell line M059J . It is possible that a mutation in ATM [38] , a protein involved in DSB repair and signaling , might partly be responsible for the extreme lack of rejoining in these cells , although the low expression of ATM does not seem to influence the radiosensitivity of M059J cells [39] . In addition , hyperPHOSphorylation of the replication protein A2 ( RPA2 ) in these cells , leading to decreased binding of RPA to ssDNA , could further influence the observed deficiency in DSB rejoining [40] The variation in the rejoining capability may also be due to species differences ; for instance the level of DNA-PKcs is much lower in hamster cells than in human cells [37,41] or there may be residual DNA-PKcs activity in the hamster mutants [41,42] . In G1-synchronized XRCC4 deficient cells , no or very little rejoining was detected up to 4 h after DSB induction , indicating that no alternative end-joining independent of DNA Ligase IV/XRRC4 occurs in the G1 phase of the cell cycle in mammalian cells .