Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein.
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 UvsY-ssDNA 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 ssDNA-dependent 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.
Bleuit
Jill S
JS
Departments of Biochemistry and Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Ma
Yujie
Y
Munro
James
J
Morrical
Scott W
SW
D000251:Adenosine Triphosphatases / Q000737:chemistry; D020816:Amino Acid Motifs; D017122:Bacteriophage T4 / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008954:Models, Biological; D009154:Mutation; D020125:Mutation, Missense; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D012965:Sodium Chloride / Q000494:pharmacology; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D004247:DNA; D000251:Adenosine Triphosphatases