DNA Metabolism in Balance: Rapid Loss of a RecA-Based Hyperrec Phenotype.

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.

Modulating cellular recombination potential through alterations in RecA structure and regulation.

The wild-type Escherichia coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to sixfold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to fourfold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50-fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of E. coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function - filament formation and the inherent DNA pairing activity of the formed filaments.

Two RecA protein types that mediate different modes of hyperrecombination.

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.

Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli.

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.

Conformational flexibility of RecA protein filament: transitions between compressed and stretched states.

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.

Characteristic thermodependence of the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The Desulfurococcus amylolyticus RadA protein (RadA(Da)) promotes recombination at temperatures approaching the DNA melting point. Here, analyzing ATPase of the RadA(Da) presynaptic complex, we described other distinguishing characteristics of RadA(Da). These include sensitivity to NaCl, preference for lengthy single-stranded DNA as a cofactor, protein activity at temperatures of over 100 degrees C, and bimodal ATPase activity. These characteristics suggest that RadA(Da) is a founding member of a new class of archaeal recombinases.

Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family.

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 ssDNA-dependent ATP hydrolysis [poly(dC) > poly(dT) > phiX174 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 degrees C than its S. cerevisiae homolog (Rad51(Sc)). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees 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 degrees C. These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site.

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.

Conjugational hyperrecombination achieved by derepressing the LexA regulon, altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12.

The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.