title: Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination.
abstract: Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination mediator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demonstrated that UvsX-H195Q/A mutants exhibit decreased ssDNA-binding affinity and altered enzymatic properties. Here, we show that unlike wild-type UvsX, the ssDNA-dependent ATPase activities of UvsX-H195Q/A are strongly inhibited by both low and high concentrations of UvsY protein. This inhibition is partially relieved by UvsY mutants with decreased ssDNA-binding affinity. The UvsX-H195Q mutant retains weak DNA strand exchange activity that is inhibited by wild-type UvsY, but stimulated by ssDNA-binding compromised UvsY mutants. These and other results support a mechanism in which the formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. Other results suggest that UvsY acts as a nucleotide exchange factor for UvsX, enhancing filament stability by increasing the lifetime of the high-affinity, ATP-bound form of the enzyme. Our findings reveal new details of the UvsX/UvsY relationship in T4 recombination, which may have parallels in other recombinase/mediator systems.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Farb
authors.forename: Scott W
authors.initials: SW
authors.lastname: Morrical
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D017122:Bacteriophage T4 / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: 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; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases