The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7.

Abstract

In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork. The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase. On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically. Both the T7 gene 2.5 protein and E. coli SSB act stoichiometrically to promote this initial strand displacement step. Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins. However, the T7 gene 2.5 protein, but not E. coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold. The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus.