A DNA binding loop in the gene 4 helicase‐primase of bacteriophage T7

Abstract

The gene 4 protein encoded by bacteriophage T7 provides both helicase and primase activities at the replication fork. The primase domain is located in the N-terminal half of the polypeptide and the helicase domain in the C-terminal half. The gene 4 protein assembles on single-stranded DNA as a hexamer and then translocates unidirectionally 5′ to 3′ along the strand using the energy of the hydrolysis of deoxythymidine tri-phosphate (dTTP), thus unwinding double-stranded DNA (dsDNA) to yield single-stranded DNA (ssDNA). The crystal structure of the helicase domain of gene 4 protein reveals a flexible segment (residues 464–475), designated as loop II, that lies between conserved motifs H3 and H4. Loop II although not having a defined motif, is distinctly basic with three lysines (K) that protrude into the central DNA-binding cavity formed by the hexameric protein. It is, therefore, a likely candidate for modulating the binding of gene 4 protein to single-stranded DNA. Using in vitro mutagenesis we have replaced K467, K471, and K473 with alanines (A). The altered protein lacking basic residues in loop II (gp4-KloopIIA) does not complement T7Δgp4 infection of E. coli. Although gp4-KloopIIA formed oligomers equally well as wild-type protein, it is severely defective in binding to ssDNA. As a consequence, ssDNA-dependent stimulation of dTTPase activity and DNA unwinding are both abolished. Thus, the basic residues of loop II are involved in the binding of gene 4 protein to single-stranded DNA. * SM and DJC contributed equally to the work.