In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling

From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260 and.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2012; 287(43):36123-31. DOI: 10.1074/jbc.M112.407825
Source: PubMed


Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently,
we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets,
p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations,
RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function,
whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment
of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially
normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

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    ABSTRACT: Activation of the replication checkpoint relies upon uncoupling of DNA polymerases and helicase activities at replication forks, which in multicellular organism results in production of long stretches of single-stranded DNA bound by the trimeric, single stranded DNA binding protein, the RPA complex. Binding of RPA to this substrate promotes synthesis of replication intermediates that contributes to checkpoint activation by allowing binding of the 9-1-1 checkpoint clamp. The RPA32kDa subunit is also phosphorylated during this process but its role in checkpoint signalling is unclear. Here we have investigated the requirement for RPA32 phosphorylation in checkpoint activation in Xenopus egg extracts. We show that phospho-deficient mutants of RPA32 stimulate checkpoint signalling at replication forks arrested with aphidicolin at both the initiation and the elongation step of DNA replication, without affecting DNA synthesis. In contrast, we show that phospho-mimetic RPA32 mutants do not stimulate checkpoint activation at unwound forks. These results indicate that the hypophosphorylated, replication fork-associated form of RPA32 functions in S-phase-dependent checkpoint signalling at unwound forks in Xenopus egg extracts while RPA32 phosphorylation may be implicated in other pathways such as repair or restart of arrested replication forks.
    No preview · Article · Oct 2012 · Biochemical and Biophysical Research Communications
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    ABSTRACT: DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.
    Preview · Article · Jan 2014 · Journal of Biological Chemistry
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    ABSTRACT: The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability.
    Full-text · Article · Mar 2014 · Genes
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