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


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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    • "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.
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    • "Whereas S4S8 phosphorylation was strongly induced after 12 and 24 h of HU treatment in shCTL, it was dramatically impaired in shPARG cells (Figure 4A, upper panel). This was correlated with the strong decrease of the slow migrating band detected with anti-RPA2 antibody and representing the hyperphosphorylated form of RPA2 (41). This strong reduction of RPA2 hyperphosphorylation in HU-treated shPARG did not result from a delayed phosphorylation, since lower levels were observed all along the release from an HU treatment of 24 h (Figure 4B). "
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    ABSTRACT: Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.
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    • "In addition, DNA damage is another key factor triggering RPA hyperphosphorylation, which is, however, distinct from the cell-cycle-dependent phosphorylation. Ionizing radiation led to RPA2 localization to DNA damage loci and RPA2 hyperphosphoryation which involves ATM and DNA-PK [30]. Bleomycin-treated cells undergoing mitosis led to RPA2 phosphorylation at Ser4/Ser8 and Thr21 which are primed by phosphorylation at Ser23 and Ser29 [33,34]. "
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    ABSTRACT: Rfa2 is a single-stranded DNA (ssDNA) binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein-protein interaction and protein-DNA interaction. In this study, we found that the Pph3/Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G1 phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3Δ or psy2Δ G1 cells exhibited diminished binding affinity to double-stranded DNA (dsDNA) but not to ssDNA. We also discovered that Cdc28 and Mec1 are responsible for Rfa2 phosphorylation in G1 phase and under DNA replication stress respectively. Moreover, mass spectrometry (MS) revealed that the domain of Rfa2 that was phosphorylated in G1 phase differed from that phosphorylated under the stress condition. Our results imply that differential phosphorylation plays a crucial role in RPA regulation.
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