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-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA 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 interactions. Therefore, we examined the effects of six different gp32 B-domain mutants 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 reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.
Villemain
J L
JL
Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-2128, USA.
Ma
Y
Y
Giedroc
D P
DP
Morrical
S W
SW
D017122:Bacteriophage T4 / Q000235:genetics* / Q000502:physiology; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004247:DNA