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PMID: 14729972 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest.

Molecular and cellular biology ·Vol. 24 ·No. 3 ·2004-02-00 ·Pages 1279-91

Davies SL, North PS, Dart A, Lakin ND, Hickson ID

Abstract

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.

MeSH Terms
Adenosine Triphosphatases/genetics,metabolism Antineoplastic Agents/pharmacology Ataxia Telangiectasia Mutated Proteins Bloom Syndrome/enzymology Cell Cycle Proteins DNA Helicases/genetics,metabolism Fibroblasts/drug effects Genetic Predisposition to Disease Humans Hydroxyurea/pharmacology Phosphorylation Phosphotransferases/metabolism Protein Serine-Threonine Kinases/metabolism RecQ Helicases S Phase/physiology Threonine/metabolism
Chemicals
Antineoplastic Agents Cell Cycle Proteins Threonine Phosphotransferases ATR protein, human Ataxia Telangiectasia Mutated Proteins Protein Serine-Threonine Kinases Adenosine Triphosphatases Bloom syndrome protein DNA Helicases RecQ Helicases Hydroxyurea
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Davies Sally L
Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.
North Phillip S
Dart Alwyn
Lakin Nicholas D
Hickson Ian D
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.24.3.1279-1291.2004
Published
2004-02-00
Pages
1279-91
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC321429
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.24.3.1279-1291.2004 PMC Full Text View journal details
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