DnaB Helicase Stimulates Primer Synthesis Activity on Short Oligonucleotide Templates

Abstract

DnaB helicase stimulated the second-order RNA primer synthesis activity of primase by over 5000-fold on DNA templates that were 23 nucleotides long. This template length is the same as the DnaB helicase thermodynamic binding site size [Jezewska, M. J., and Bujalowski, W. (1996) Biochemistry 35, 2117-2128]. This phenomenal stimulation was achieved by increasing the template affinity of primase by over 300-fold and increasing the catalytic rate by over 15-fold. It was necessary to determine the optimal amount of DnaB helicase to achieve this stimulation because helicase stimulation was cooperative at low concentration and inhibitory at high helicase concentration. The cooperative stimulation at low concentration indicated the presence of a time-dependent assembly step that preceded the active state. Besides stimulating primase activity, DnaB helicase also prevented primase from synthesizing RNA primers that were longer than the template sequence. In the absence of DnaB helicase, the majority of primers synthesized by primase were longer than the template and were named "overlong primers" [Swart, J. R., and Griep, M. A. (1995) Biochemistry 34, 16097-16106]. In contrast, the helicase-stimulated RNA primers were from 10 to 14 nucleotides in length with the 12-mer representing the majority of the total RNA primers produced. It was shown that DnaB helicase stabilized the open or single-stranded conformation of the template, which favored the synthesis of the template-length-dependent primers. In contrast, when primase acted alone, it stabilized the 3'-end hairpin conformation of the template so that the template's 3'-hydroxyl served as a "DNA primer" from which primase elongated to create the overlong primers.

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

“…1, lanes 1 and 5, respectively). It has been shown that "overlong primers" occurred when the 3Ј-end of the template forms a hairpin such that the terminal nucleotide joins with another portion of the template through the base pair (27). In the presence of DnaB helicase, primase demonstrated remarkable primer synthesis from both CTG-template ( Fig.…”

“…1-It has been demonstrated that, at the replication fork, DnaB recruits primase via a protein-protein interaction, which consequently produces primer (26,27). However, their interaction is assumed to be transient since there is no direct evidence for it.…”

“…Kinetic analysis suggests that E. coli primase, acting alone, is the slowest RNA polymerase, with an in vitro rate of about one primer every thousand seconds (26). However, the activity of primase on oligonucleotide templates could be stimulated upon the addition of DnaB helicase at an optimum ratio (27). This indicates that the interaction of DnaB with primase stimulates the rate of the primer synthesis.…”

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

“…The E. coli DnaG gene product is the model eubacterial primase because its structure and function have been extensively characterized. It has been demonstrated that the E. coli primase is slow (9), has low-fidelity (9), binds G4-ori ssDNA as a dimer (10,11), and that DnaB helicase stimulates its catalytic activity over 15-fold (12 Recently, it was demonstrated that the primases and replication fork helicases from mesophilic S. aureus and thermophilic Geobacillus stearothermophilus have properties that diverge from their E. coli homologs (14,15). Both primases initiated from 5'-TTA-3' and 5'-CTA-3'.…”

“…6C lines Effect of helicase on primase initiation specificity. When E. coli primase activity was stimulated by its replicative helicase, it initiated from all trinucleotides that were tested (12,13). To determine whether S. aureus DnaC also relaxed the initiation specificity of its primase, the d(TTA), d(CTT), d(CTG), or d(TTT) templates (2 μM) were incubated with primase (400 nM) and DnaC (133 nM hexamer) for 30 min (Fig.…”