title: 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.affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
authors.forename: Angela M
authors.initials: AM
authors.lastname: Fan
authors.forename: Linghua
authors.initials: L
authors.lastname: Meng
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Doe
authors.forename: Phillip S
authors.initials: PS
authors.lastname: North
authors.forename: Ian D
authors.initials: ID
authors.lastname: Hickson
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D021581:Active Transport, Cell Nucleus; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D001816:Bloom Syndrome / Q000235:genetics / Q000378:metabolism; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004351:Drug Resistance; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D000071156:Promyelocytic Leukemia Protein; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D053484:RecQ Helicases; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin