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 (DnaG(BA)) 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 DnaG(BA) binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaG(BA) bound to the origin of bacteriophage G4 (G4ori) with approximately 8-fold higher affinity. DnaG(BA) was strongly stimulated (>or=75-fold) by its cognate helicase, DnaB(BA), during RNA primer synthesis. With the G4ori ssDNA template, DnaG(BA) formed short (<or=20 nucleotides) primers in the absence of DnaB(BA). The presence of DnaB(BA) increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaB(BA) is thus indicative of a positive effector role for DnaB(BA). By contrast, Escherichia coli DnaB helicase (DnaB(EC)) did not stimulate DnaG(BA) and inhibited primer synthesis to near completion. This observed effect of E. coli DnaB(EC) is indicative of a strong negative effector role for heterologous DnaB(EC). We conclude that DnaG(BA) 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 (DnaB(BA)) acted as a positive effector of primer synthesis.
Biswas
Subhasis B
SB
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA. subhasis.biswas@umdnj.edu
Wydra
Eric
E
Biswas
Esther E
EE
D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D019915:DNA Primase