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Free Content Allosteric regulation of the primase (DnaG) activity by the clamp-loader (τ) in vitro

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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 τ subunit of the clamp-loader that loads the β clamp and interconnects the core polymerases on the leading and lagging strands. The τ–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 τ subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB–τ interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V τ mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of τ elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native τ protein. Thus changes in the τ–DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of τ are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by τ-interacting components of the replisome through the τ–DnaB−DnaG pathway.
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Document Type: Research Article

Affiliations: 1: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK. 2: Department of Microbiology and Pathology, 986495 University of Nebraska Medical Center, Omaha, Nebraska 68198–6495, USA. 3: Department of Chemistry, University of Nebraska-Lincoln, Nebraska 68588–0304, USA.

Publication date: April 1, 2009

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