Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides.
Bacteriophage T4 provides an important model for the biochemistry and genetics of DNA metabolism. Phage-encoded proteins conduct all essential steps of T4 DNA replication, repair, and recombination. Central to these three processes is the T4 UvsX protein, a member of the filamentous, ATP-dependent class of general recombination enzymes typified by the Escherichia coli RecA protein. Like RecA, UvsX forms presynaptic filaments on single-stranded (ss) DNA, which are the obligatory nucleoprotein intermediates in recombination. Aspects of the T4 presynaptic filament are explored by quantitative characterization of the UvsX-ssDNA interaction using an etheno-derivitized single-stranded DNA molecule, epsilonDNA, whose fluorescence is enhanced by UvsX binding. Studies with this model lattice show that UvsX exhibits a moderate level of cooperativity (omega=100) when binding to epsilonDNA with a binding-site size (n) equal to four nucleotide residues. Salt-stability studies of this complex reveal that the non-hydrolyzable ATP analog, ATPgammaS, induces a high-affinity binding mode that is distinguishable from complexes formed with ADP or in the absence of a nucleotide cofactor. With this new information, both functional relationships between the UvsX and RecA recombinases, and implications for UvsX interactions with the other proteins of the T4 presynaptic filament (UvsY and gp32) may be further explored.
Ando
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RA
Department of Biochemistry Department of Microbiology and Molecular Genetics, and Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Morrical
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SW
D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005453:Fluorescence; D019426:Integrases; D008565:Membrane Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D045522:Recombinases; D012492:Salts / Q000494:pharmacology; D017186:Titrimetry; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009711:Nucleotides; D045522:Recombinases; D012492:Salts; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1