RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations.

Using the Force The mention of dark quenching may conjure up images of light-saber battles between the forces of ruddy-faced Good and mellifluous-voiced Evil. In the molecular sciences, however, the scenario is usually far more benign and significantly more practical. The process of dark quenching, in which bringing two molecules together, a fluor and a quencher, results in the abrogation of a light signal, can be seen as being the flip-side of fluorescence resonance energy transfer (FRET). FRET assays, where the close proximity of two molecules creates fluorescence, have been used previously to monitor, in real-time, the process of homologous recombination in vitro. A challenge with this and other fluorescence assays is to obtain a sufficiently high signal over the background, an issue that commonly confounds many such experiments. Kaboev et al. on p. 736 of this issue therefore decided to go over to the dark side by using a dsDNA template carrying a FAM fluor as well as a DABCYL quencher molecule. The propinquity of the fluor and quencher mean that no signal is seen until recombination takes place, at which time one of the DNA strands is liberated and the inhibition is released, resulting in a fluorescent signal. Using this modification, as well as designing the probe with a triple repeat of the target sequence, the authors were able to demonstrate an impressive 5- to 10-fold improvement in the signal-to-background ratio.

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...

Different conformational states of the filaments formed by RecA protein from a radiation resistant strain Deinococcus radiodurance (RecADr) in solution were investigated using small angle neutron scattering. Scattering by the protein self-polymer was consistent with a long helix model, with the pitch of the helix being lower than that in the crystal structure. Compared to those of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, helical filaments of RecA from D. radiodurance exhibited a lower helical pitch and lower stability at low Mg2+ concentrations or under conditions of elevated ionic strength in the absence of ATP (adenosine triphosphate). Formation of an active filament upon binding of ATPγS and either single- or double-stranded DNA brought about a significant increase in the helix pitch and a moderate decrease in the cross-sectional gyration radius, but resulted in little change in the number of monomers per helix turn. The helix pitch value of the RecADr presynaptic complex was conservative and close to that found for other RecA proteins and their analogs.