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Borgstahl, GEO,Brader, K,Mosel, A,Liu, SQ,Kremmer, E,Goettsch, KA,Kolar, C,Nasheuer, HP,Oakley, GG
2014
September
Dna Repair
Interplay of DNA damage and cell cycle signaling at the level of human replication protein A
Published
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Homologous recombination DNA double-stranded breaks DNA damage response DNA repair Phosphorylation Isoelectric focusing G2 phase STRAND BREAK REPAIR HOMOLOGOUS RECOMBINATION ATAXIA-TELANGIECTASIA INDUCED HYPERPHOSPHORYLATION INDUCED PHOSPHORYLATION BINDING-PROTEIN S-PHASE SACCHAROMYCES-CEREVISIAE RPA PHOSPHORYLATION DISTINCT ROLES
21
12
23
Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphoiylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphoiylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites. (C) 2014 Elsevier B.V. All rights reserved.
10.1016/j.dnarep.2014.05.005
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