Allosteric regulation of the primase (DnaG) activity by the clamp-loader (tau) in vitro.
During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the tau subunit of the clamp-loader that loads the beta clamp and interconnects the core polymerases on the leading and lagging strands. The tau-DnaB-DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the tau subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB-tau interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V tau mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of tau elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native tau protein. Thus changes in the tau-DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of tau are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by tau-interacting components of the replisome through the tau-DnaB-DnaG pathway.
Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, UK.
Kiran
K
Chintakayala
Cristina
C
Machón
Anna
A
Haroniti
Marilyn A
MA
Larson
Steven H
SH
Hinrichs
Mark A
MA
Griep
Panos
P
Soultanas
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D053488:DnaB Helicases / Q000378:metabolism*; D015723:Gene Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D019915:DNA Primase; D053488:DnaB Helicases