From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold

Bochkarev, A.; Bochkareva, Elena

doi:10.1016/j.sbi.2004.01.001

Cited by 215 publications

(221 citation statements)

References 41 publications

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“…Furthermore, there is evidence suggesting that the ssDNA binding properties of hRPA and scRPA differ in detail (48). Finally, there seems to be no consensus on the site sizes that characterize any proposed different binding modes (27,50,51).One difficulty in assessing whether the range of RPA binding site sizes reported reflect true differences is that these studies used a variety of different approaches and were performed under different solution conditions. Since the E. coli SSB tetramer in complex with ssDNA has been shown to undergo a salt dependent binding mode transition (39,52), it is possible that some of the variability observed for the scRPA binding site size is due to differences in the solution conditions used.…”

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confidence: 99%

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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confidence: 99%

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

2006

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“…In their normal context, the constellation of BRC repeats presumably promotes HDR; however, cellular expression of individual BRC repeats recapitulates phenotypes of Brca2 mutant cells, including reduced HDR, implying that by themselves they interfere with Rad51 function (12)(13)(14). C-terminal to the BRC repeats is a region that binds ssDNA (15,16). This region consists of four globular domains, including two oligonucleotide͞oligosaccharide-binding (OB) folds, which have close structural homology to two OB folds in replication protein A (RPA) 70, the largest subunit of the ssDNA-binding protein RPA (15), and an unusual tower domain appended to one of the globular domains (16).…”

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confidence: 99%

“…C-terminal to the BRC repeats is a region that binds ssDNA (15,16). This region consists of four globular domains, including two oligonucleotide͞oligosaccharide-binding (OB) folds, which have close structural homology to two OB folds in replication protein A (RPA) 70, the largest subunit of the ssDNA-binding protein RPA (15), and an unusual tower domain appended to one of the globular domains (16). The extreme C terminus of BRCA2 contains an additional Rad51-binding motif (11), which is distinct from the BRC repeats, and which has been shown recently to undergo regulated Rad51 binding in response to CDK phosphorylation (17).…”

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confidence: 99%

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

Saeki

Siaud

Christ

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.double-strand break ͉ mammalian cells ͉ Rad51 ͉ homologous recombination ͉ BRC repeat

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“…ssDNA-Binding Proteins ssDNA-binding proteins, or SSBs, are ubiquitous proteins that play fundamental roles in DNA recombination, replication, and repair, as well as in DNA damage signaling, cell-cycle control, and regulation of gene expression (Wold 1997;Lohman et al 1988;Lohman and Ferrari 1994;Binz et al 2004;Bochkarev and Bochkareva 2004;Richard et al 2009). Well-characterized examples include E. coli SSB protein, bacteriophage T4 Gp32 protein, and the RPA proteins of yeast and humans (Table 1).…”

Section: Properties Of Dna-pairing Proteinsmentioning

confidence: 99%

“…Common properties of SSBs include stoichiometric, sequence nonspecific binding to ssDNA, highaffinity and (typically) cooperative binding to ssDNA, weak or nonexistent affinity for dsDNA, and the ability to stimulate cognate replication and recombination enzymes. SSBs all contain one or more copies of the oligonucleotide/oligosaccharide-binding (OB) fold motif (Arcus 2002;Bochkarev and Bochkareva 2004). SSB sequences are divergent, however, and their tertiary structures vary dramatically between species.…”

Section: Properties Of Dna-pairing Proteinsmentioning

confidence: 99%

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

Morrical

2015

Cold Spring Harb Perspect Biol

116

127

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The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and singlestrand annealing pathways-DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.

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From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold

Cited by 215 publications

References 41 publications

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

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

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

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