TI - Extensive ssDNA end formation at DNA double strand breaks in non-homologous end-joining deficient cells during the S phase . AB - Background Efficient and correct repair of DNA damage , especially DNA double strand breaks , is critical for cellular survival . Defects in the DNA repair may lead to cell death or genomic instability and development of cancer . Non-homologous end-joining ( NHEJ ) is the major repair pathway for DNA double strand breaks in mammalian cells . The ability of other repair pathways , such as homologous recombination , to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood . Results In this report we demonstrate that long single-stranded DNA ( ssDNA ) ends are formed at radiation-induced DNA double strand breaks in NHEJ deficient cells . At repair times >= 1 h , processing of unrejoined DNA double strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase ( DNA-PK ) or DNA Ligase IV/XRCC4 . The ssDNA formation was cell cycle dependent , since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells . Furthermore , in wild type cells irradiated in the presence of DNA-PKcs ( catalytic subunit of DNA-PK ) inhibitors or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autoPHOSphorylation sites ( ABCDE ) , no ssDNA was formed . The ssDNA generation also greatly influences DNA double strand break quantification by pulsed-field gel electrophoresis , resulting in overestimation of the DNA double strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA ( i e , lysis at 50degC ) are used . Conclusion We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double strand breaks prevents the formation of long ssDNA ends at double strand breaks during the S phase , indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required .