SummaryThe replacement of Escherichia coli recA gene (recA Ec protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.
The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51 Pa ) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes. We cloned the RAD51 gene from the cDNA library of the thermotolerant P. angusta strain BKM Y1397. Induction of this gene in a rad51-deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation. Purified Rad51 Pa protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51 Pa per 3 nucleotides) and DNA specificity for ssDNAdependent ATP hydrolysis [poly(dC) > poly(dT) > X174 ssDNA > poly(dA) > double-stranded M13 DNA]. An inefficient ATPase and very low cooperativity for ATP interaction position Rad51 Pa closer to Rad51 than to RecA. Judging by thermoinactivation, Rad51 Pa alone was 20-fold more thermostable at 37°C than its S. cerevisiae homolog (Rad51 Sc ). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54°C, whereas Rad51 Sc was completely inactive at 47°C. A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51 Pa occurred only at temperatures above 42°C. These reaction characteristics suggest that Rad51 Pa is dependent on high temperatures for activity.Pichia angusta, one of the species of the taxonomic complex Hansenula polymorpha (25), is a methylotrophic yeast which has attracted considerable attention as a promising host for the production of recombinant proteins because of its powerful promoter elements, its ability to grow at high density on inexpensive substrates, and the unusual properties of its homologous and nonhomologous recombination systems promoting a multiple tandem integration of a nonlinearized plasmid in the host chromosome (for reviews, see references 11, 28, and 31). One additional characteristic that might have a biotechnological advantage is its thermotolerance, an ability to grow at temperatures (42°C and higher) which are not acceptable for other yeast strains.Among 2,500 P. angusta novel protein-encoding genes now identified, 6% have no homologs in Saccharomyces cerevisiae (3). Little is known about the recombination or DNA repair proteins in P. angusta, such as the homologous DNA transferase Rad51 and its paralogs. This recombinase is a RecARadA-like protein, found in all three domains of life (4), which forms filaments on single-stranded DNA (ssDNA) in the presence of ATP (and thus possesses ATPase activity) and promotes homologous pairing and strand exchange, which are the two main steps in the initiation of homologous recombination and recombination repair, as well (6,16,27). All members of the RecA-RadA-Rad51 recombinase superfamily form nucleoprotein filaments of similar structures and stoichiometries, and they display similar preferences in DNA substrates for DNAdependent ATP hy...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.