title: ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage.
abstract: The cell cycle checkpoint kinase Chk1 is phosphorylated and activated by ATR in response to DNA damage and is crucial for initiating the DNA damage response. A number of factors act in concert with ATR to facilitate Chk1 phosphorylation, including Rad17-RFC, the Rad9-Rad1-Hus1 complex, TopBP1 and Claspin. Rad17 is required for loading of Rad9-Rad1-Hus1 (9-1-1) onto sites of DNA damage. Although phosphorylation of Rad17 by ATR is required for checkpoint function, how this affects 9-1-1 regulation remains unclear. We report that exposure of cells to DNA damage or replication stress results in Rad17-dependent immobilisation of Rad9 into nuclear foci. Furthermore, expression of mutant Rad17 that cannot be phosphorylated by ATR (Rad17(AA)), or downregulation of ATR, results in a decreased number of cells that display Rad9 foci. Photobleaching experiments reveal an increase in the dynamic behaviour of Rad9 within remaining foci in the absence of ATR or following expression of Rad17(AA). Together, these data suggest a model in which Rad17 and ATR collaborate in regulating Rad9 localisation and association at sites of DNA damage.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QD, UK.
authors.forename: Annette L
authors.initials: AL
authors.lastname: Medhurst
authors.forename: Daniël O
authors.initials: DO
authors.lastname: Warmerdam
authors.forename: Ildem
authors.initials: I
authors.lastname: Akerman
authors.forename: Edward H
authors.initials: EH
authors.lastname: Verwayen
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.forename: Veronique A J
authors.initials: VA
authors.lastname: Smits
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Lakin
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; C069930:rad9 protein; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D017346:Protein Serine-Threonine Kinases