Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA

Bochkarev, A.; Pfuetzner, Richard A.; Edwards, A.M.; Frappier, Lori

doi:10.1038/385176a0

Cited by 541 publications

(665 citation statements)

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“…2 Replication protein A (RPA), a component of the origin recognition complex, is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. 3,4 Moreover, RPA is involved in DNA recombination and repair. [5][6][7][8] Additionally, it has also been implicated in the regulation of apoptosis and gene expression.…”

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Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

et al. 2007

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Replication protein A (RPA), a component of the origin recognition complex, is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. Experimental studies in colon cancer cell lines have shown that RPA protein may be the target of cytotoxins designed to inhibit cellular proliferation. This is the first study to investigate the expression of RPA1 and RPA2 subunits of RPA protein and assess their prognostic value in colon cancer patients. We analyzed immunohistochemically the expression of RPA1 and RPA2 proteins in a series of 130 colon cancer resection specimens in relation to conventional clinicopathological parameters and patients' survival. Statistical significant positive associations emerged between: (a) RPA1 and RPA2 protein expressions (P ¼ 0.0001), (b) RPA1 and RPA2 labelling indices (LIs) and advanced stage of the disease (P ¼ 0.001 and 0.003, respectively), (c) RPA1 and RPA2 LIs and the presence of lymph node metastasis (P ¼ 0.002 and 0.004, respectively), (d) RPA1 LI and the number of infiltrated lymph nodes (P ¼ 0.021), (e) RPA2 LI and histological grade of carcinomas (P ¼ 0.05). Moreover, a statistical significant higher RPA1 LI was observed in the metastatic sites compared to the original ones (P ¼ 0.012). RPA1 and RPA2 protein expression associated with adverse patients' outcome in both univariate (log rank test: Po0.00001 and 0.00001, respectively) and multivariate (Cox model: P ¼ 0.092 and 0.0001, respectively) statistical analysis. Statistical significant differences according to the expression of RPA1 and RPA2 proteins were also noticed in the survival of stage II (Po0.00001 and 0.0016, respectively) and stage III (P ¼ 0.0029 and 0.0079, respectively) patients. In conclusion, RPA1 and RPA2 proteins appear to be useful prognostic indicators in colon cancer patients and attractive therapeutic targets for regulation by tumor suppressors or other proteins involved in the control of cell proliferation. Modern Pathology (2007) 20, 159-166.

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Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

et al. 2007

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“…Whereas T4 gene 32 protein contains one OBfold, the E. coli SSB tetramer contains four OB-folds and the hetero-trimeric RPA contains six OB-folds. Each OB-fold within RPA could potentially provide a site for ssDNA binding (21)(22)(23)(24). Four OB-folds are located within RPA70, referred to as F, A, B and C, (see Figure 1), a fifth OB-fold (D) is contained within the RPA32 subunit, and a sixth OB-fold (E) is contained within RPA14.…”

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Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

2006

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We have examined the single stranded DNA binding properties of the S. cerevisiae Replication Protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry and sedimentation equilibrium in order to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants and binding enthalpy of scRPA-((dT) L ) complexes as a function of oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is stable heterotrimer over the range of [NaCl] examined (0.02 M to 1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n=18−20 nucleotides at low [NaCl] to n=26 −28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0°C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT) L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in occluded site size. Measurements of the ΔH obs for scRPA binding to (dT) L at 1.5 M NaCl, yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 ± 1 nucleotides), stabilized at low [NaCl], in which only three of its OB-folds are used, and a higher site size mode (n = 27 ± 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found either under any conditions examined. Thus, scRPA shows some similar behavior to the E. coli SSB homo-tetramer, which also shows binding mode transitions, but some significant differences also exist.Single stranded (ss) DNA binding (SSB) proteins exist in nearly all organisms and play central roles in all aspects of DNA metabolism, including DNA replication, repair, and recombination. However, the structural forms of SSB proteins differ considerably among different organisms. For example, the bacteriophage T4 gene 32 protein, the first such SSB protein identified (1, 2) is a monomer, whereas the E. coli SSB protein, is a homotetramer (3,4), as are most SSB proteins from eubacteria. In contrast, the eukaryotic SSB protein, commonly called Replication Protein A (RPA) is a hetero-trimer (5,6).In all eukaryotes, RPA consists of three highly conserved subunits of approximately 70, 32, and 14 kDa, named according to their respective molecular weights, and all three subunits are required for function in vivo (5,6). However, RPA homologues display distinct species specificity, such that scRPA from yeast cannot substitute for human RPA (hsRPA) Figure showing the results of agarose gel electrophoresis experiments at low (20 mM) and high (1.5 M) NaCl concentrations for scRPA-ssDNA complexes formed at different protein to DNA ratios indicating low or no cooperativity upon formation of these complexes. This material is availa...

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“…Structurally, RPA is composed of multiple homologous domains classified as oligonucleotide/oligosaccharide binding (OB) folds (11). The bulk of RPA's DNA binding activity has been attributed to two OB folds present in the central region of RPA-p70.…”

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“…The bulk of RPA's DNA binding activity has been attributed to two OB folds present in the central region of RPA-p70. These two DNA binding domains are termed DBD A and B and the structure of which has been solved by X-ray crystallography in the presence and absence of DNA (11,12). Evidence also suggests that the central domain of RPA-p34 and the C-terminal domain of RPA-p70, DBDs D and C, respectively, may contribute to DNA binding affinity (13).…”

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DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage

et al. 2005

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Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cellcycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.Replication protein A (RPA) 1 is the major eukaryotic single-stranded DNA (ssDNA) binding protein. RPA is a heterotrimeric protein composed of 70, 34, and 14 kDa subunits and was discovered as an essential component of the SV40 cell free DNA replication system (1, 2). RPA's role in DNA replication is to bind and stabilize ssDNA and to stimulate DNA polymerase α (3). Central roles have also been discovered in nucleotide excision repair, DNA mismatch repair, DNA recombination, and the nonhomologous end joining pathway for repair of DNA double strand breaks (4-8). RPA-coated ssDNA also appears to be a key structure for the activation of checkpoint signaling in response to DSBs and stalled DNA † This work was supported by Public Health Service Grant CA82741 and Grant CDMRP OC020223 to J.J.T. and Public Health Service Grants NS34782 and ES06096 and a research grant from the A-T Children's Project to K.D. 1 Abbreviations: RPA, replication protein A; XPA, xeroderma pigmentosum group A protein; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; CIP, calf intestinal phosphatase; PAGE, poly-acrylamide gel electrophoresis; OB, oligonucleotide/ oligosaccharide binding; DBD, DNA binding domain; CHCA, α-cyano-4-hydroxy cinnamic acid. (9,10). In all of these pathways, RPA binds to single-stranded regions of DNA and interacts with a variety of proteins that ultimately govern how genetic information is copied, repaired, and maintained. NIH Public AccessStructurally, RPA is composed of multiple homologous domains classified as oligonucleotide/oligosaccharide binding (OB) folds (11). The bulk of RPA's DNA binding activity has been attributed to two OB folds present in the central region of RPA-p70. These two DNA binding domains are termed DBD A and B and the structure of which has been solved by X-ray crystallography in the presence and absence of DNA (11,12). Evidence also suggests that the central domain of RPA-p34 and the C-terminal domain of RPA-p70, DBDs D and C, res...

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Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA

Cited by 541 publications

References 20 publications

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage

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