Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis.
Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical cross-linker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.
Mitkova
Atanaska V
AV
Department of Molecular Biology, School of Osteopathic Medicine, University of Medicine & Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Khopde
Sujata M
SM
Biswas
Subhasis B
SB
D016880:Anisotropy; D001426:Bacterial Proteins; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry*; D004277:DNA, Single-Stranded; D053488:DnaB Helicases; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005456:Fluorescent Dyes; D005976:Glutaral / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry*
D001426:Bacterial Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012313:RNA; D019915:DNA Primase; D004265:DNA Helicases; D053488:DnaB Helicases; D005976:Glutaral