A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer

Abstract

The bacteriophage T7 gene 4 protein, like a number of helicases, is believed to function as a hexamer. The amino acid sequence of the T7 gene 4 protein from residue 475 to 491 is conserved in the homologous proteins of the related phages T3 and SP6. In addition, part of this region is conserved in DNA helicases such as Escherichia coli DnaB protein and phage T4 gp41. Mutations within this region of the T7 gene 4 protein can reduce the ability of the protein to form hexamers. The His475 → Ala and Asp485 → Gly mutant proteins show decreases in nucleotide hydrolysis, single-stranded DNA binding, double-stranded DNA unwinding, and primer synthesis in proportion to their ability to form hexamers. The mutation Arg487 → Ala has little effect on oligomerization, but nucleotide hydrolysis by this mutant protein is inhibited by single-stranded DNA, and it has a higher affinity for dTTP, suggesting that this protein is defective in the protein-protein interactions required for efficient nucleotide hydrolysis and translocation on single- stranded DNA. Gene 4 protein can form hexamers in the absence of a nucleotide, but dTTP increases hexamer formation, as does dTDP, to a lesser extent, demonstrating that the protein self-association affinity is influenced by the nucleotide bound. Together, the data demonstrate that this region of the gene 4 protein is important for the protein-protein contacts necessary for both hexamer formation and the interactions between the subunits of the hexamer required for coordinated nucleotide hydrolysis, translocation on single-stranded DNA, and unwinding of double-stranded DNA. The fact that the gene 4 proteins form dimers, but not monomers, even while hexamer formation is severely diminished by some of the mutations, suggests that the proteins associate in a manner with two separate and distinct protein-protein interfaces.