Formation of D loops by the UvsX protein of T4 bacteriophage: a comparison of the reaction catalyzed in the presence or absence of gene 32 protein

Abstract

The UvsX protein of T4 bacteriophage will catalyze the formation of D loops between linear single-stranded DNA (ssDNA) and homologous supercoiled double-stranded DNA (dsDNA) in the absence of T4 gene 32 protein (gp32). This reaction requires one monomer of UvsX protein per three nucleotides of ssDNA so that the ssDNA is completely covered with UvsX protein. Under these conditions, high rates of ATP hydrolysis are observed, and one-third of the products are joined paranemically. The reaction proceeds through a mechanism that creates homology-independent coaggregates of UvsX protein, dsDNA, and ssDNA. When UvsX protein is added to only 1 monomer per 8 nucleotides, but with 1 monomer of gp32 per 12 nucleotides, the rate of ATP hydrolysis is depressed, but D-loop formation is enhanced. Nearly all of the product is bound in plectonemic joints, and no coaggregated intermediates are formed. Coaggregate formation at high concentrations of UvsX protein is not inhibited by the presence of gp32; gp32 simply allows for efficient formation of D loops at such low concentrations of UvsX protein that coaggregates are not constructed. Electron microscopic visualization of the joint structures in this reaction reveals that both gp32 and UvsX protein are bound to the ssDNA. The single-stranded DNA binding (SSB) protein of Escherichia coli will substitute only partially for gp32: in the presence of SSB protein, D-loop formation can be catalyzed at one UvsX protein monomer per eight nucleotides, and it is accomplished without the formation of coaggregates, but a major portion of the product is joined paranemically.