Abstract
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.
Highlights
DNA double-strand breaks (DSBs) can be caused by exogenous and endogenous mechanisms, such as ionizing radiation, reactive oxygen species, or replication fork collapse [1,2,3,4,5]
Reliance on homologous recombination (HR) should be advantageous for cells to preserve genome integrity and replication-associated DSBs appear to be preferentially repaired by HR [2,3]
To address the relative contribution of HR and nonhomologous end-joining (NHEJ) to DSB repair of human cells, we generated a series of knockout mutant cell lines deficient for DSB repair factors by using the Nalm-6 cell line, in which we have recently developed a system that enables rapid production of knockout mutants by gene targeting [12,13]
Summary
DNA double-strand breaks (DSBs) can be caused by exogenous and endogenous mechanisms, such as ionizing radiation, reactive oxygen species, or replication fork collapse [1,2,3,4,5]. HR allows for accurate repair of DSBs with the use of homologous DNA sequences [2,3], whereas NHEJ repairs broken DNA ends with little or no homology and is often associated with nucleotide loss [4,5]. Because of such intrinsic differences in accuracy between HR and NHEJ, the two pathways should differentially contribute to repair of, and cellular survival after, different types of DSBs. Apparently, reliance on HR should be advantageous for cells to preserve genome integrity and replication-associated DSBs appear to be preferentially repaired by HR [2,3]. More recent studies show that initiation of end-resection by CtIP and the MRN complex is one of the key mechanisms that influence the usage of HR and NHEJ upon DSBs [9,10,11]
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