21 Single-Stranded DNA Binding Proteins

Kowalczykowski, Stephen C.; Bear, David G.; Hippel, Peter H. von

doi:10.1016/s1874-6047(08)60347-9

Cited by 109 publications

(80 citation statements)

References 173 publications

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“…The nucleotide concentrations of poly(dT), ssDNA, and dsDNA were measured using extinction coefficients of 7.3 ϫ 10 3 , 8.1 ϫ 10 3 , and 6.5 ϫ 10 3 M Ϫ1 cm Ϫ1 , respectively. Rad51 and Rad52 proteins (14), RPA (19), and E. coli SSB protein (20,21) were prepared as described. Rabbit polyclonal antibody against RPA was obtained from W. D. Heyer (University of California, Davis).…”

Section: Methodsmentioning

confidence: 99%

Rad52 Protein Associates with Replication Protein A (RPA)-Single-stranded DNA to Accelerate Rad51-mediated Displacement of RPA and Presynaptic Complex Formation

Sugiyama

Kowalczykowski

2002

Journal of Biological Chemistry

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214

198

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The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.The Rad52 epistasis group of proteins, including Rad51 protein, Rad52 protein, and replication protein A (RPA), 1 are important for both mitotic and meiotic recombination, matingtype switching, and repair of DNA double strand breaks in Saccharomyces cerevisiae. Rad51 protein, which is a homologue of the Escherichia coli RecA protein (1-3), is conserved in a wide variety of eukaryotic organisms from yeast to humans (4). Like RecA protein, Rad51 protein binds single-stranded DNA (ssDNA) to form the functional presynaptic complex, which mediates DNA strand exchange (5). However, unlike RecA protein, Rad51 protein readily binds double-stranded DNA (dsDNA), and the binding to dsDNA strongly inhibits DNA strand exchange (6). Therefore, not unexpectedly, the binding of Rad51 protein to dsDNA present as secondary structure in ssDNA severely limits DNA strand exchange (7). For this reason, Rad51 protein-mediated DNA strand exchange depends strongly on a ssDNA-binding protein to eliminate DNA secondary structure.RPA, which is a heterotrimeric ssDNA-binding protein (8, 9), greatly stimulates DNA strand exchange by Rad51 protein, provided that RPA is added to a preexisting complex of Rad51 protein and ssDNA (5). However, RPA will inhibit DNA strand exchange when it is allowed to bind ssDNA before Rad51 protein. Previously, we offered an interpretation for this dichotomous role of RPA in Rad51 protein-mediated DNA strand exchange (7). According to this view, RPA aids DNA strand exchange by disrupting DNA secondary structure, which is an impediment to presynaptic complex formation. However, because RPA and Rad51 protein both compete for these same ssDNA binding sites, RPA can also be an impediment to presynaptic complex formation. Nevertheless, when the molecular ratios of Rad51 protein and RPA to ssDNA are appropriate (2-3 nucleotides per Rad51 protein and 10 -20 nucleotides per RPA), the steady-state product of this competitive process is a uniform Rad51 protein-ssDNA complex with little DNA secondary structure. Based on this model, after disruption of DNA secondary structure, RPA is expected to be displaced by Rad51 protein. Previo...

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Section: Methodsmentioning

confidence: 99%

Rad52 Protein Associates with Replication Protein A (RPA)-Single-stranded DNA to Accelerate Rad51-mediated Displacement of RPA and Presynaptic Complex Formation

Sugiyama

Kowalczykowski

2002

Journal of Biological Chemistry

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214

198

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“…For the NLLS analysis of protein-DNA complexes, σ DNA was fixed to the value determined independently for DNA, while floating for σ protein-DNA complexes. The values of for the protein-DNA complexes were calculated as the weight-average sum of for the individual components, as in equation : (2) where is the partial specific volume for the protein-DNA complex, M Rf and M Df are molecular masses of scRPA and the DNA substrate (g mol −1 ), and and are the partial specific volumes of the scRPA and DNA substrate respectively (ml g −1 ). The sedimentation velocity experiments were analyzed using the program DCDT + (version 1.16, John S. Philo) to obtain estimates of the molecular masses.…”

Section: Analytical Ultracentrifugationmentioning

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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“…We have designated the genes responsible for this behavior SOC8-I and SOC8-2, suppressors of cdc8. These plasmids transform at a lower efficiency than the CDC8-containing plasmids (see Table 1 (14).…”

Section: Resultsmentioning

confidence: 99%

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

Kuo¹,

Campbell²

1983

Mol. Cell. Biol.

163

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The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp5O bearing the yeast ARSI, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to.grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product. The CDC8 protein of Saccharomyces cerevisiae is essential for chromosomal replication in vivo and is required for cell division. When the temperature-sensitive cdc8 mutant is grown at the permissive temperature, 23°C, and then shifted to the restrictive temperature, 36°C, cells accumulate that contain a nucleus located at the isthmus between parent cell and bud, but which do not divide. The first wave of DNA synthesis after shift up does not occur (8). The product of the CDC8 gene is apparently required throughout the period of DNA synthesis, since synchronized cultures of cells defective in the gene cease nuclear DNA replication when shifted to 36°C within the S period (9). Mitochondrial DNA replication also ceases at the nonpermissive temperature in cdc8 mutants (22), and replication of the 2-,um circle plasmid is defective in cdc8 mutants (17).Recently the CDC8 protein has been purified to homogeneity, using in vitro replication systems (1, 15). The purified protein binds to singlestranded DNA and stimulates DNA polymerase I activity on single-stranded DNA templates. The CDC8 protein may be identical to protein C, discovered by Chang et al. (4).To carry out detailed biochemical and functional characterization of an enzyme involved in DNA replication, it is necessary to obtain large quantities of purified protein. In Escherichia coli, one way to overcome the problem of low yield of replication proteins is to clone the gene coding for a given protein into temperatureinducible bacteriophage lambda vectors or into a high-copy-number plasmid. Overproduction of the replication proteins DNA polymerase I (12), DNA ligase (23), and dnaC protein (13) has been achieved. Overproduction of the LEU2 gene product in yeast strains carrying the LEU2 gene on an autonomously replicating, high-copy-number plasmid has also been achieved (10). Therefore, the same approach used in E. coli is applicable in yeast.In this co...

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21 Single-Stranded DNA Binding Proteins

Cited by 109 publications

References 173 publications

Rad52 Protein Associates with Replication Protein A (RPA)-Single-stranded DNA to Accelerate Rad51-mediated Displacement of RPA and Presynaptic Complex Formation

Rad52 Protein Associates with Replication Protein A (RPA)-Single-stranded DNA to Accelerate Rad51-mediated Displacement of RPA and Presynaptic Complex Formation

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

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

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