A DOMAIN OF THE GENE-4 HELICASE-PRIMASE OF BACTERIOPHAGE-T7 REQUIRED FOR THE FORMATION OF AN ACTIVE HEXAMER

The bacteriophage T7 gene 4 protein, like a number of helicases, is be lieved to function as a hexamer, The amino acid sequence of the T7 gen e 4 protein from residue 475 to 491 is conserved in the homologous pro teins of the related phages T3 and SP6. In addition, part of this regi on is conserved in DNA helicases such as Escherichia coli DnaB protein and phage T4 gp41. Mutations within this region of the T7 gene 4 prot ein can reduce the ability of the protein to form hexamers, The His(47 5)-->Ala and Asp(485)-->Gly mutant proteins show decreases in nucleoti de hydrolysis, single-stranded DNA binding, double-stranded DNA unwind ing, and primer synthesis in proportion to their ability to form hexam ers, The mutation Arg(487)-->Ala has little effect on oligomerization, but nucleotide hydrolysis by this mutant protein is inhibited by sing le stranded DNA, and it has a higher affinity for dTTP, suggesting tha t this protein is defective in the protein-protein interactions requir ed for efficient nucleotide hydrolysis and translocation on single-str anded DNA, Gene 4 protein can form hexamers in the absence of a nucleo tide, but dTTP increases hexamer formation, as does dTDP, to a lesser extent, demonstrating that the protein self-association affinity is in fluenced 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 be tween the subunits of the hexamer required for coordinated nucleotide hydrolysis, translocation on single stranded DNA, and unwinding of dou ble-stranded DNA. The fact that the gene 4 proteins form dimers, but n ot monomers, even while hexamer formation is severely diminished by so me of the mutations, suggests that the proteins associate in a manner with two separate and distinct protein-protein interfaces.