Title | Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery. |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Murphy AK, Fitzgerald M, Ro T, Kim JHyun, Rabinowitsch AI, Chowdhury D, Schildkraut CL, Borowiec JA |
Journal | J Cell Biol |
Volume | 206 |
Issue | 4 |
Pagination | 493-507 |
Date Published | 2014 Aug 18 |
ISSN | 1540-8140 |
Keywords | Ataxia Telangiectasia Mutated Proteins, BRCA2 Protein, Camptothecin, Cell Line, Tumor, Chromatin, Cyclin-Dependent Kinase 2, DNA Damage, DNA Repair, DNA Replication, DNA, Single-Stranded, DNA-Binding Proteins, Fanconi Anemia Complementation Group N Protein, Humans, Hydroxyurea, Multiprotein Complexes, Nuclear Proteins, Nucleic Acid Synthesis Inhibitors, Phosphoprotein Phosphatases, Phosphorylation, Poly(ADP-ribose) Polymerase Inhibitors, Replication Protein A, RNA Interference, RNA, Small Interfering, Topoisomerase I Inhibitors, Tumor Suppressor Proteins |
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. |
DOI | 10.1083/jcb.201404111 |
Alternate Journal | J. Cell Biol. |
PubMed ID | 25113031 |
PubMed Central ID | PMC4137056 |
Grant List | R01 GM083185 / GM / NIGMS NIH HHS / United States P30 CA016087 / CA / NCI NIH HHS / United States P30CA16087 / CA / NCI NIH HHS / United States R01CA142698 / CA / NCI NIH HHS / United States 5R01 GM045751 / GM / NIGMS NIH HHS / United States R01 CA142698 / CA / NCI NIH HHS / United States R01 GM045751 / GM / NIGMS NIH HHS / United States |
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