Repair pathway of DSBs induced by ionizing radiation is distinctly different from that of DSBs that arise during DNA replication

Various types of DNA lesions are generated continuously in the cells. Some lesions could block a DNA replication fork leading to a daughter strand gap and double-strand break (DSB). Studies of E. Coli and yeast indicate that daughter strand gap is filled by translesion synthesis (TLS) polymerases, and repaired by homologous recombination (HR). DSBs are also induced by IR, which is widely used to study DSB repair pathways in mammals. Previous studies have shown that there are two major DSB repair pathways, nonhomologous end joining (NHEJ) and HR. We have made from chicken B lymphocyte line DT40 the following gene disrupted clones: mutants of HR (rad51, mre11, rad54), NHEJ (ku70), and TLS (rev3), and obtained three conclusions from the following findings. (1) Conditional inactivation of rad51 and Mre11 causes extensive chromosomal breaks and kill the cells. In contrast, ku70 shows virtually no spontaneous chromosomal breaks, and grows normally. Thus, HR plays a major role in repairing DSBs that occur during replication. (2) The rad54 mutant, where HR capability is marginally impaired, can grow nearly normal kinetics but exhibit IR sensitivity at late S to G2 phase, while ku70 exhibit IR sensitivity particularly at G1 to early S phase. Cells deficient in both genes (rad54/ku70) exhibit extremely high IR sensitivity in any phases. These observations suggest that NHEJ plays a critical role in DSB repair following IR while HR can repair only chromatid breaks by facilitating recombination with the intact sister. (3) We found that rad54/rev3 is synthetic lethal and exhibit extensive chromosomal breaks before cell death. Presumably, HR and TLS play a substantially overlapping role in processing daughter strand gaps caused by replication block. In summary, HR and TLS may prevent chromosomal breaks during replication while NHEJ is a key player in repairing IR induced DSBs in higher eukaryotic cells