Abstract
BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.
Highlights
BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure
We propose that the absence of BLM leads to accumulation of replicationassociated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by nonhomologous end-joining (NHEJ)
Using an I-SceIbased DSB repair assay system designed in this study, we found that BLMϪ/Ϫ cells have an NHEJ activity similar to that of wild type
Summary
BLM؊/؊ cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression These phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. LIG4Ϫ/Ϫ cells displayed aberrant end-joining events such as frequent nucleotide loss and rearrangements, which were much more pronounced in a BLMϪ/Ϫ background These results suggest the existence of an alternative end-joining pathway that is independent of LIG4 and highly error prone. Based on these results, we propose that BLM deficiency leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells when repaired by NHEJ. Our results suggest the interdependence of the endjoining pathway and the function of BLM helicase
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