are unable to join 3' overhangs that require gap filling, but remain proficient for gap filling at 5' overhangs. This is because the primer terminus used to initiate DNA synthesis is less stable at 3' overhangs, necessitating a specialized NHEJ polymerase. Ligation The DNA ligase IV complex, consisting of the catalytic subunit DNA ligase IV and its cofactor XRCC4 (Dnl4 and Lif1 in yeast), performs the ligation step of repair. XLF, also known as Cernunnos, is homologous to yeast Nej1 and is also required for NHEJ. While the precise role of XLF is unknown, it interacts with the XRCC4/DNA ligase IV complex and likely participates in the ligation step. Recent evidence suggests that XLF promotes re-adenylation of DNA ligase IV after ligation, recharging the ligase and allowing it to catalyze a second ligation. Other In yeast, Sir2 was originally identified as an NHEJ protein, but is now known to be required for NHEJ only because it is required for the transcription of Nej1. NHEJ and heat-labile sites Induction of heat-labile sites (HLS) is a signature of ionizing radiation. The DNA clustered damage sites consist of different types of DNA lesions. Some of these lesions are not prompt DSBs but they convert to DSB after heating. HLS are not evolved to DSB under physiological temperature (370 C). Also, the interaction of HLS with other lesions and their role in living cells is yet elusive. The repair mechanisms of these sites are not fully revealed. The NHEJ is the dominant DNA repair pathway throughout the cell cycle. The DNA-PKcs protein is the critical element in the center of NHEJ. Using DNA-PKcs KO cell lines or inhibition of DNA-PKcs does not affect the repair capacity of HLS. Also blocking both HR and NHEJ repair pathways by dactolisib (NVP-BEZ235) inhibitor showed that repair of HLS is not dependent on HR and NHEJ. These results showed that the repair mechanism of HLS is independent of NHEJ and HR pathways Regulation The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is