Single-stranded DNA binding properties of the uvsY recombination protein of bacteriophage T4.
The uvsY protein is an essential component of the bacteriophage T4 general recombination machinery. The properties of this 16 kDa protein include selective binding to ssDNA, as well as specific protein-protein interactions with other T4 recombination proteins including uvsX (general recombinase) and gp32 (ssDNA-binding protein). uvsY promotes the assembly of uvsX-ssDNA filaments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA. To better understand the role of uvsY in the T4 recombination system, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model single-stranded polynucleotide, epsilonDNA, which is a fluorescent, etheno-modified form of random-sequence ssDNA. We have found that the binding of uvsY protein enhances the fluorescence of the epsilonDNA lattice and that the maximal amount of fluorescence enhancement observed is dependent on salt concentration. In addition, we have used the epsilonDNA fluorescence enhancement assay to establish thermodynamic parameters of binding and to define some of the molecular details of uvsY-epsilonDNA interactions. We show that uvsY binds to epsilonDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to epsilonDNA over unmodified ssDNA. The significance of these results is discussed in light of current models of uvsY action in the T4 recombination system.
Sweezy
M A
MA
Department of Biochemistry, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405, USA.
Morrical
S W
SW
D017104:Bacteriophage M13 / Q000235:genetics; D017122:Bacteriophage T4 / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011995:Recombination, Genetic; D019346:Sodium Acetate / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; D012965:Sodium Chloride; D019346:Sodium Acetate