Relationship between Hexamerization and ssDNA Binding Affinity in the uvsY Recombination Protein of Bacteriophage T4

Ando, Richard A.; Morrical, Scott W.

doi:10.1021/bi991917h

Cited by 9 publications

(14 citation statements)

References 46 publications

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“…Most of these genes were first identified by the isolation of amber or temperature-sensitive conditional-lethal mutations and were assigned numbers (Table 2) before their functions were determined (264). 5,6,7,8,9,10,11,12,15,18,19,25,26,27,28,29,48,53 a Genes in parentheses appear in another, primary category. Primary functional assignments and the corresponding colors are used in Fig.…”

Section: Characterized T4 Genes and The Early Geneticsmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

704

801

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SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

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Section: Characterized T4 Genes and The Early Geneticsmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

704

801

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“…9b). In addition, both Rad52 protein and UvsY protein form ring-like structure (heptamer for Rad52 protein and hexamer for UvsY), and this multimerization is required for UvsY function (11,38,39). Therefore, for the reasons outlined above, we favor the facilitated nucleation model as depicted in Fig.…”

Section: Displacement Of Rpa From Ssdna Is Limited By Nucleation Of Amentioning

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

216

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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“…Conversely, a truncated form of UvsY protein lacking the COOH-terminal 36 amino acid residues sediments as a 2.1 S monomer at all salt concentrations examined (24). The self-association properties of missense mutants UvsY K58A and UvsY K58A,R60A were examined by sedimentation velocity.…”

Section: Expression and Purification Of Uvsymentioning

confidence: 99%

“…Limited chymotrypsinolysis digests the 137-amino acid UvsY protein into two putative domains: an aminoterminal fragment or domain containing the first 101 residues, and a carboxyl-terminal fragment or domain containing the remaining 36 residues (13). The NH 2 -terminal fragment retains weak ssDNA binding activity, but is completely deficient in self-association (hexamerization) and in UvsY-Gp32 and UvsY-UvsX interactions (13,24). The decrease in ssDNA binding affinity appears to result from the loss of intrahexamer synergism or cooperativity in this monomeric form of UvsY, not from disruption of the ssDNA binding site itself (24).…”

mentioning

confidence: 97%

“…The NH 2 -terminal fragment retains weak ssDNA binding activity, but is completely deficient in self-association (hexamerization) and in UvsY-Gp32 and UvsY-UvsX interactions (13,24). The decrease in ssDNA binding affinity appears to result from the loss of intrahexamer synergism or cooperativity in this monomeric form of UvsY, not from disruption of the ssDNA binding site itself (24). Despite the loss of protein-protein contacts, the NH 2 -terminal fragment of UvsY retains the ability to stimulate the ssDNA-dependent ATPase and DNA strand exchange activities of UvsX, but at dramatically lower levels than full-length UvsY (13).…”

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confidence: 99%

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Mutations in a Conserved Motif Inhibit Single-stranded DNA Binding and Recombination Mediator Activities of Bacteriophage T4 UvsY Protein

Bleuit

Munro

et al. 2004

Journal of Biological Chemistry

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The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsYssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNAdependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.

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Relationship between Hexamerization and ssDNA Binding Affinity in the uvsY Recombination Protein of Bacteriophage T4

Cited by 9 publications

References 46 publications

Bacteriophage T4 Genome

Bacteriophage T4 Genome

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

Mutations in a Conserved Motif Inhibit Single-stranded DNA Binding and Recombination Mediator Activities of Bacteriophage T4 UvsY Protein

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