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Molecular and Cellular Biology, October 2005, p. 8925-8937, Vol. 25, No. 20
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.20.8925-8937.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phosphorylation of BLM, Dissociation from Topoisomerase III{alpha}, and Colocalization with {gamma}-H2AX after Topoisomerase I-Induced Replication Damage

V. Ashutosh Rao,1 Angela M. Fan,1 LingHua Meng,1 Christopher F. Doe,1 Phillip S. North,2 Ian D. Hickson,2 and Yves Pommier1*

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255,1 Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom2

Received 21 June 2005/ Returned for modification 28 June 2005/ Accepted 20 July 2005

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 Top3{alpha}. 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 Top3{alpha} 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.

* Corresponding author. Mailing address: National Cancer Institute, NIH, 37 Convent Drive, Building 37, Room 5068, Bethesda, MD 20892-4255. Phone: (301) 496-5944. Fax: (301) 402-0752. E-mail: pommier{at}nih.gov.

Molecular and Cellular Biology, October 2005, p. 8925-8937, Vol. 25, No. 20
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.20.8925-8937.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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