Replication protein A: Directing traffic at the intersection of replication and repair

College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska 68583, USA.
Frontiers in Bioscience (Impact Factor: 3.52). 06/2010; 15(3):883-900. DOI: 10.2741/3652
Source: PubMed


Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic processes.

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Available from: Greg G Oakley
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    • "B), a recognized DSB marker (Oakley and Patrick, 2010). Overall, ATR activation in our experimental conditions does not directly mirror the extent of replication interference, nor the amount of ssDNA detected at RIs (Table 1), and likely reflects yet-undefined signaling determinants that escape systematic cell-based and single-molecule analyses. "
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    ABSTRACT: Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy. © 2015 Zellweger et al.
    Full-text · Article · Mar 2015 · The Journal of Cell Biology
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    • "In chromatinbound RPA2, Ser33 is heavily phosphorylated in response to DNA damage, markedly contrasting with the cytosolic RPA2 pattern. Ser33 is thought to be phosphorylated by ATR [3] and although ATR is present in the cytosol [67], it does not appear to be acting upon cytosolic RPA2 in response to damage, as it does for chromatinbound RPA2. This confirms the requirement of ATRIP recognition of RPA-ssDNA complex for activation of ATR phosphorylation of RPA- NT [22]. "
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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.
    Full-text · Article · Sep 2014 · DNA Repair
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    • "Next we investigate whether, similarly to GSK3B, also GSK3A inhibition influences DNA damage response/repair systems. To this end we analysed γH2AX foci formation, as a marker of the DNA damage response [19], and RPA70 foci formation, as a marker of DNA repair [20], in HCT116p53KO cells treated with 5FU. As in the case of GSK3B, also blocking GSK3A, either by silencing (Fig. 4A, B) or by use of a specific inhibitor (Fig. 4C, D) did not affect DNA damage sensing (Fig. 4A, C) as indicated by the formation of γH2AX-positive foci. "
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    ABSTRACT: Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.
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