Phosphorylation Of Blm, Dissociation From Topoisomerase Iii Alpha, And Colocalization With Gamma-H2ax After Topoisomerase I-Induced Replication Damage
MOLECULAR AND CELLULAR BIOLOGY(2005)
Abstract
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 III alpha (Top3 alpha) 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 Top3a. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (gamma-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3 alpha, 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 gamma-H2AX but not with Top3a or PML. Thus, BLM appears to dissociate from Top3 alpha and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in gamma-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.
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Key words
phosphorylation,nuclear proteins,dna repair,dna binding proteins,signal transduction,histones,dna replication,cell line,transcription factors,dna damage,drug resistance
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