DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates

Abstract

The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.