DNA-binding properties of T4 UvsY recombination mediator protein: polynucleotide wrapping promotes high-affinity binding to single-stranded DNA.
To carry out homologous recombination events in the cell, recombination proteins must be able to recognize and form presynaptic filaments on single-stranded DNA (ssDNA) in the presence of a vast excess of double-stranded DNA (dsDNA). Therefore recombination machineries stringently discriminate between ssDNA and dsDNA lattices. Recent single-molecule studies of bacteriophage T4 recombination proteins revealed that, surprisingly, the UvsY recombination mediator protein binds stronger to stretched dsDNA molecules than to stretched ssDNA. Here, we show that for relaxed DNA lattices, the opposite is true: UvsY exhibits a 1000-fold intrinsic affinity preference for ssDNA over dsDNA at moderate salt concentrations. This finding suggests that UvsY preferentially loads UvsX recombinase onto ssDNA under physiological conditions. The biochemical basis for high-affinity UvsY-ssDNA binding was investigated by hydrodynamic and cross-linking methods. Results show that UvsY forms ring-like hexamers in solution, and that ssDNA binds to multiple subunits within each hexamer, consistent with ssDNA wrapping. The data support a model in which ssDNA wrapping by UvsY protein is important for the selective nucleation of presynaptic filaments on ssDNA versus dsDNA, and for the coordinated transfer of ssDNA from Gp32 (SSB) to UvsY (RMP) to UvsX (recombinase) during filament assembly.
Xu
Hang
H
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Beernink
Hans T H
HT
Morrical
Scott W
SW
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; D004247:DNA