Phosphorylation of BLM, dissociation from topoisomerase IIIalpha, and colocalization with gamma-H2AX after topoisomerase I-induced replication damage.

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 IIIalpha (Top3alpha) 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 Top3alpha. 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, Top3alpha, 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 Top3alpha or PML. Thus, BLM appears to dissociate from Top3alpha 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.
Authors	V Ashutosh Rao, Angela M Fan, Linghua Meng, Christopher F Doe, Phillip S North, Ian D Hickson, Yves Pommier
Journal	Molecular and cellular biology (Mol Cell Biol) Vol. 25 Issue 20 Pg. 8925-37 (Oct 2005) ISSN: 0270-7306 [Print] United States
PMID	16199871 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References	Cell Cycle Proteins DNA-Binding Proteins H2AX protein, human Histones Neoplasm Proteins Nuclear Proteins Promyelocytic Leukemia Protein Transcription Factors Tumor Suppressor Proteins PML protein, human ATM protein, human ATR protein, human Ataxia Telangiectasia Mutated Proteins Protein Serine-Threonine Kinases Adenosine Triphosphatases Bloom syndrome protein DNA Helicases RecQ Helicases DNA Topoisomerases, Type I Camptothecin
Topics	Active Transport, Cell Nucleus Adenosine Triphosphatases (chemistry, deficiency, genetics, metabolism) Ataxia Telangiectasia Mutated Proteins Bloom Syndrome (genetics, metabolism) Camptothecin (pharmacology) Cell Cycle Proteins (metabolism) Cell Line DNA Damage DNA Helicases (chemistry, deficiency, genetics, metabolism) DNA Repair DNA Replication DNA Topoisomerases, Type I (metabolism) DNA-Binding Proteins (metabolism) Drug Resistance Histones (metabolism) Humans Models, Biological Neoplasm Proteins (metabolism) Nuclear Proteins (metabolism) Phosphorylation Promyelocytic Leukemia Protein Protein Serine-Threonine Kinases (metabolism) RecQ Helicases Signal Transduction Transcription Factors (metabolism) Tumor Suppressor Proteins (metabolism)

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