Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis

Cell Cycle Control Laboratory, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland.
Nucleic Acids Research (Impact Factor: 9.11). 09/2009; 37(18):6028-41. DOI: 10.1093/nar/gkp605
Source: PubMed

ABSTRACT The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.

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Available from: Heinz Peter Nasheuer, Jul 08, 2015
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    • "Also a putative role for RPA in the export of nuclear mRNA has been described [69]. Another possibility, given RPA's exclusion from chromatin during mitosis [13], is that the cytosol could be a type of storage facility for RPA; however that does not explain the difference in phosphorylation patterns on cytosolic S and G2 RPA2. Although we have observed that RPA is present in the cytosol and regulated via protein phosphorylation in response to DNA damage in a cell cycle dependent manner, RPA's role in the cytosol remains to be determined. "
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    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 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 phosphorylation 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 phosphorylation 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.
    DNA Repair 09/2014; 21:12–23. DOI:10.1016/j.dnarep.2014.05.005 · 3.36 Impact Factor
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    • "However, RPA also mediates the recruitment of the ATR/ATRIP complex to the single stranded regions and initiates in this way the DDR signaling cascades, which among others inhibit cell cycle progression through activation of the corresponding checkpoints (Cimprich & Cortez, 2008; Zegerman & Diffley, 2009). Indeed, there is evidence that RPA functions as a checkpoint activator (Stephan et al., 2009), as well as a regulator of the repair process, possibly through shifts in its function by DNA damage-mediated post-translational modifications (Anantha et al., 2007; Vassin et al., 2009). RPA also facilitates indirectly Rad51 filament formation by mediating DNA-Rad52 or DNA-BRCA2 interactions (see below) (Mortensen et al., 2009; Thorslund & West, 2007). "
    DNA Repair - On the Pathways to Fixing DNA Damage and Errors, 09/2011; , ISBN: 978-953-307-649-2
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    • "Although early studies gave conflicting results on whether DNA-PKcs phosphorylates RPA in response to DNA damage (Boubnov et al., 1995; Fried et al., 1996), the bulk of the evidence is consistent with such a role (Shao et al., 1999; Wang et al., 2001; Block et al., 2004; Cruet-Hennequart et al., 2006; Anantha et al., 2007; Cruet-Hennequart et al., 2008; Stephan et al., 2009). DNA-PKcs also regulates the stability of histone mRNA abundance through phosphorylation of the RNA helicase UPF1, linking DNA-PKcs to histone synthesis and thus DNA replication (Kaygun and Marzluff, 2005; Muller et al., 2007). "
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    ABSTRACT: High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks. Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their roles in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku, DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its role in fork restart. Most cancer therapeutic strategies create DNA replication stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.
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