Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stra nded DNA (ssDNA)-binding protein required for phage DNA replication and rec ombination, gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by D NA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA intera ctions. Therefore, we examined the effects of six different gp32 B-domain m utants on T4 in vitro systems for DNA synthesis and homologous pairing, We find that the B-domain is essential for gp32's stimulation of these reactio ns. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e, wild-type gp3 2 approximate to R4K > K3A approximate to R4Q > R4T > R4G much greater than gp32-B. However, the functional defect of a particular mutant is often gre ater than can be explained simply by its ability to saturate the ssDNA at e quilibrium, suggesting additional defects in the proper assembly and activi ty of DNA polymerase and recombinase complexes on ssDNA, which may derive f rom a decreased lifetime of gp32-ssDNA clusters.