title: Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress.
abstract: The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32kDa subunit (RPA32), and the APC-RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and γ-H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and γ-H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle.
authors.affiliation: Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia. mariana.brocardo@sydney.edu.au
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Brocardo
authors.forename: James A
authors.initials: JA
authors.lastname: Borowiec
authors.forename: Beric R
authors.initials: BR
authors.lastname: Henderson
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D046911:Macromolecular Substances / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D013312:Stress, Physiological; D015854:Up-Regulation
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D018797:Cell Cycle Proteins; D002843:Chromatin; D006657:Histones; D046911:Macromolecular Substances; D019384:Nucleic Acid Synthesis Inhibitors; D010446:Peptide Fragments; D051716:Replication Protein A; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C516794:RPA2 protein, human; D006918:Hydroxyurea