Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

Abstract

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

“…Previous studies have also shown that genetic disruption or kinase inhibition of DNA-PKcs, or genetic disruption of other c-NHEJ factors, cause an increase in the frequency of SSA (24,52); whereas disruption of another component of the MRE11-RAD50-NBS1 complex (NBS1) causes a decrease in SSA (37,65). As SSA requires extensive end processing to reveal the homology that flanks the DSB, these findings support the notion that c-NHEJ factors, including DNA-PKcs, are important to limit such extensive DSB end processing, whereas RAD50 and NBS1 promote this process.…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Errorprone repair of the DSB by single strand annealing (SSA) is increased in BRCA2 mutants [87,88]-a pattern reminiscent of HR mutants in yeast and in other vertebrate cells. In contrast, SSA is partly dependent on BRCA1, suggesting that BRCA1 also participates in an earlier step in HR, perhaps working with the MRN complex to control processing of the DSB end [88,89]. These studies do not assay HR at stalled forks, since the induced DSB has no relation to replication arrest.…”

MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks

“…MRX, the yeast orthologue of MRN, functions during NHEJ in the budding yeast Saccharomyces cerevisiae but not in the fi ssion yeast Schizosaccharomyces pombe ( 27 -29 ). In mammalian cells, MRN is not recruited to the site of DSBs generated by the I-sceI endonuclease in G1-phase cells, and MRN does not appear to be required for the NHEJmediated repair of these DSBs ( 30,31 ).…”