Abstract
Abstract 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 DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-stranded break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phase of the cell cycle and all three subunits of RPA contain phosphorylation sites. The exact set of HR-relevant phosphorylation sites of RPA is unknown. In this study, the phosphorylation sites of chromatin-bound RPA in S and G2 phase were identified. After DNA damage, phosphorylation included pSer4/8, pSer12, pThr21, pSer23 and pSer33 of RPA2. Phosphorylation of these sites increased with time after DNA damage. Using a general ATM/ATR phosphorylation antibody, only RPA2 had substantial pSer/pThr ATM/ATR signals. Additionally, pTyr was detected on RPA1 and RPA2 and was removed in response to DSBs, indicating the regulatory action of a phosphatase. A capillary isoelectric focusing immunoassay used under native conditions revealed in high resolution isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Strikingly, RPA is a phosphoprotein in control G2 cell lysates with up to 5 putative phospho-isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33 and RPA1 pTyr. DNA damaged lysates contained 9 isoforms, containing up to 14 phosphates. DNA damage isoforms contained RPA2 pSer4/8, pSer12, pThr21, pSer23, pSer33 and other not yet identified phosphorylation sites. Citation Format: Kerry Brader, Adam Mosel, Shengqin Liu, Elizabeth Kremmer, Kaitlin Goettsch, Heinz-Peter Nasheuer, Greg Oakley, Gloria Borgstahl. Interplay of DNA damage and cell cycle signaling on Replication Protein A in human cells. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 33. doi:10.1158/1538-7445.CANSUSC14-33
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
