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 essenti al steps of T4 DNA replication, repair, and recombination. Central to these three processes is the T4 UvsX protein, a member of the filament ous, ATP-dependent class of general recombination enzymes typified by the Escherichia coli RecA protein, Like RecA, UvsX forms presynaptic f ilaments on single-stranded (ss) DNA, which are the obligatory nucleop rotein intermediates in recombination. Aspects of the T4 presynaptic f ilament are explored by quantitative characterization of the UvsX-ssDN A interaction using an etheno-derivitized single-stranded DNA molecule , epsilon DNA, whose fluorescence is enhanced by UvsX binding. Studies with this model lattice show that UvsX exhibits a moderate level of c ooperativity (omega =100) when binding to epsilon DNA with a binding-s ite size (n) equal to four nucleotide residues. Salt-stability studies of this complex reveal that the non-hydrolyzable ATP analog, ATP gamm a S, 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 wit h the other proteins of the T4 presynaptic filament (UvsY and gp32) ma y be further explored. (C) 1998 Academic Press.