TI - DNA Ligase IV/XRCC4 recruitment by DNA-PK to DSBs is necessary to prevent the formation of ssDNA at the ends . AB - To test the availability of DSB ends for single strand processing when fast and efficient rejoining of DSBs by NHEJ is not possible , the kinase activity of DNA-PKcs was inhibited by wortmannin or NU7026 . The inhibition of autoPHOSphorylation prevents the release of DNA-PKcs from the DNA [10,29] and hence the rejoining of DSBs . In M059K cells and V3 cells complemented with wt DNA-PKcs , which were irradiated in the presence of wortmannin or NU7026 , 70% or more of the DNA fragments were still unrejoined after 4 h ( Fig 4A ) compared to lt 10% in non-inhibited cells (Fig 1A) . However , the number of DNA fragments detected did not differ between the two DNA extraction protocols , in contrast to the findings in cells lacking DNA-PK such as M059J and V3 . The lack of ssDNA formation in DNA-PKcs inhibited cells was confirmed by immunofluorescence detection of BrdU . After 4 h of repair no increase in BrdU staining , corresponding to ssDNA , was detected in M059K cells incubated with NU7026 , as compared to that in cells not incubated with the DNA-PK kinase inhibitor (Fig 4B) . The formation of ssDNA appears to be dependent on the absence of DNA-PK at the DSB ends ; inhibition of the kinase activity , and consequently inhibition of the rejoining of DSBs by NHEJ , is not sufficient for generation of ssDNA ends . Further evidence that DNA-PKcs protect DSB ends from resection was provided in V3 cells ( DNA-PKcs deficient ) transfected with a plasmid containing a mutated DNA-PKcs in six of its autoPHOSphorylation sites (ABCDE) [6,30] . The ABCDE mutant cells have normal DNA-PKcs kinase activity but are radiosensitive , as a result of defective NHEJ , and the lack of autoPHOSphorylation at the ABCDE sites blocks the ends from processing and ligation [6 , 7] . When we irradiated V3 cells expressing this ABCDE mutant form of DNA-PKcs and extracted DNA with the warm or cold protocol after different repair times , no significant difference in the number of DNA fragments detected by PFGE was seen ( Fig 4C ) . These results support the data showing that DNA-PKcs protects the ends from resection when present at the ends , even though it is catalytically inactive . However , when the activity of DNA-PKcs was inhibited in the XRCC4 deficient cell line GM16147 , ssDNA was still formed at repair times >= 1 h , as detected by DNA extraction with the warm and cold protocols ( Fig 5A ) . In accordance with these PFGE results , the majority of the irradiated GM16147 cells with incorporated BrdU and incubated with NU7026 had a higher ( II ) or much higher level ( III ) of ssDNA 4 h after irradiation , compared to control cells ( Fig 5B and 5C ) . These data strongly suggest that it is the recruitment of Ligase IV/XRCC4 to the ends by DNA-PK that is the important factor for end protection ; in NU7026 or wortmannin treated cells Ligase IV/XRCC4 can still bind to DSB ends , since the kinase activity of DNA-PKcs is dispensable for the binding of Ligase IV/XRCC4 to the ends [10] . Ligase IV/XRCC4 apparently prevents the generation of ssDNA ends even though ligation of the ends by NHEJ is inhibited .