Modulating cellular recombination potential through alterations in RecA structure and regulation

Abstract

SummaryThe 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 SSBcoated 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.

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

“…The variations are sometimes subtle and sometimes not [19, 84–87]. The same theme is highlighted in recent work demonstrating substantial improvements in the capacity of some mutant variants of E. coli RecA protein to promote conjugational recombination [88]. Most important, the variations do not reflect an evolutionary approach to some common and most efficient platform for DNA pairing and strand exchange.…”

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

“…Some RecA mutant proteins are less inhibited by RecX protein. There is only a slight decrease in the rate of ATP hydrolysis of the RecA4C17 protein or RecAD112R protein at high RecX concentration [14,15]. Previously we showed that RecX and SSB act as functional antagonists; i.e., the inhibitory effect of RecX on RecA decreases substantially in the presence of SSB [16].…”

Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes

“…The frequency of meiotic recombination in the aw-wv segment in the F 1 RecA plants is 46.7%, which is 1.2 times higher than in the corresponding non transgenic hybrids (39.6%) ( Table 6). However, like in the case of the wv d segment, the rf value for the aw wv region does not exceed the rf values in the non transgenic Marglob × Mo938 hybrids (Tables 4 and 6).…”

“…It has become known recently that the deletion of several amino acid residues of the C end of the E. coli RecA protein leads to a 4.3 fold enhancement of its activity [46]. It is possible that the fusion of RecA at the C end with the lichenase reporter protein LicBM3 in the hybrid protein NLS RecA LicBM3 caused a decay of its activity and resulted in the absence of any effect in the F 1 NLS RecA LicBM3 plants.…”