Allosteric regulation of the primase (DnaG) activity by the clamp-loader (τ) 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 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.

Protein Hydrolysates Are Avoided by Herbivores but Not by Omnivores in Two-Choice Preference Tests

Background: The negative sensory properties of casein hydrolysates (HC) often limit their usage in products intended for human consumption, despite HC being nutritious and having many functional benefits. Recent, but taxonomically limited, evidence suggests that other animals also avoid consuming HC when alternatives exist. Methodology/Principal Findings: We evaluated ingestive responses of five herbivorous species (guinea pig, mountain beaver, gopher, vole, and rabbit) and five omnivorous species (rat, coyote, house mouse, white-footed mouse, and deer mouse; N = 16–18/species) using solid foods containing 20% HC in a series of two-choice preference tests that used a nonprotein, cellulose-based alternative. Individuals were also tested with collagen hydrolysate (gelatin; GE) to determine whether it would induce similar ingestive responses to those induced by HC. Despite HC and GE having very different nutritional and sensory qualities, both hydrolysates produced similar preference score patterns. We found that the herbivores generally avoided the hydrolysates while the omnivores consumed them at similar levels to the cellulose diet or, more rarely, preferred them (HC by the white-footed mouse; GE by the rat). Follow-up preference tests pairing HC and the nutritionally equivalent intact casein (C) were performed on the three mouse species and the guinea pigs. For the mice, mean HC preference scores were lower in the HC v C compared to the HC v Cel tests, indicating that HC’s sensory qualities negatively affected its consumption. However, responses were species-specific. For the guinea pigs, repeated exposure to HC or C (4.7-h sessions; N = 10) were found to increase subsequent HC preference scores in an HC v C preference test, which was interpreted in the light of conservative foraging strategies thought to typify herbivores. Conclusions/Significance: This is the first empirical study of dietary niche-related taxonomic differences in ingestive responses to protein hydrolysates using multiple species under comparable conditions. Our results provide a basis for future work in sensory, physiological, and behavioral mechanisms of hydrolysate avoidance and on the potential use of hydrolysates for pest management.