Phosphorylation of BLM Dissociation from Topoisomerase IIIα and Colocalization with γ-H2AX after Topoisomerase I-Induced Replication Damage

摘要

Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIα (Top3α) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3α. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (γ-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3α, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with γ-H2AX but not with Top3α or PML. Thus, BLM appears to dissociate from Top3α and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in γ-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.