TY - JOUR
T1 - Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery
AU - Murphy, Anar K.
AU - Fitzgerald, Michael
AU - Ro, Teresa
AU - Kim, Jee Hyun
AU - Rabinowitsch, Ariana I.
AU - Chowdhury, Dipanjan
AU - Schildkraut, Carl L.
AU - Borowiec, James A.
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84906511112&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84906511112&partnerID=8YFLogxK
U2 - 10.1083/jcb.201404111
DO - 10.1083/jcb.201404111
M3 - Article
C2 - 25113031
AN - SCOPUS:84906511112
SN - 0021-9525
VL - 206
SP - 493
EP - 507
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 4
ER -