title: Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.
abstract: Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016.
authors.forename: Anar K
authors.initials: AK
authors.lastname: Murphy
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016.
authors.forename: Michael
authors.initials: M
authors.lastname: Fitzgerald
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016.
authors.forename: Teresa
authors.initials: T
authors.lastname: Ro
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016.
authors.forename: Jee Hyun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016.
authors.forename: Ariana I
authors.initials: AI
authors.lastname: Rabinowitsch
authors.affiliation: Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02215.
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Chowdhury
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Carl L
authors.initials: CL
authors.lastname: Schildkraut
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University School of Medicine, New York, NY 10016 James.Borowiec@nyumc.org.
authors.forename: James A
authors.initials: JA
authors.lastname: Borowiec
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D024682:BRCA2 Protein / Q000235:genetics; D002166:Camptothecin / Q000494:pharmacology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics; D051357:Cyclin-Dependent Kinase 2; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D000076164:Fanconi Anemia Complementation Group N Protein; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D034622:RNA Interference; D034741:RNA, Small Interfering; D051716:Replication Protein A / Q000235:genetics* / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D002843:Chromatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000076164:Fanconi Anemia Complementation Group N Protein; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; C512790:PALB2 protein, human; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D059004:Topoisomerase I Inhibitors; D025521:Tumor Suppressor Proteins; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; C516794:RPA2 protein, human; D010749:Phosphoprotein Phosphatases; C080406:protein phosphatase 4; D006918:Hydroxyurea; D002166:Camptothecin
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