title: Purification and characterization of the T4 bacteriophage uvsX protein.
abstract: Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells. Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis. Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur. J. Biochem. 148, 127-134). At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J. Biol. Chem. 260, 4484-4491). We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products. Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers. The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis. We present a method that allows the purification of the uvsX protein to essential homogeneity. We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E. coli host protein of 32,000 daltons whose gene is unknown. The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D011995:Recombination, Genetic; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D000251:Adenosine Triphosphatases