Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis. The mechanism of action of B. anthracis DNA primase (DnaGBA) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaGBA binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaGBA bound to the origin of bacteriophage G4 (G4ori) with approximately 8-fold higher affinity. DnaGBA was strongly stimulated (≥75-fold) by its cognate helicase, DnaBBA, during RNA primer synthesis. With the G4ori ssDNA template, DnaGBA formed short (≤20 nucleotides) primers in the absence of DnaBBA. The presence of DnaBBA increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaBBA is thus indicative of a positive effector role for DnaBBA. By contrast, Escherichia coli DnaB helicase (DnaBEC) did not stimulate DnaGBA and inhibited primer synthesis to near completion. This observed effect of E. coli DnaBEC is indicative of a strong negative effector role for heterologous DnaBEC. We conclude that DnaGBA is capable of interacting with DnaB proteins from both B. anthracis and E. coli; however, between DnaB proteins derived from these two organisms, only the homologous DNA helicase (DnaBBA) acted as a positive effector of primer synthesis.