[Deinococcus radiodurans RecX and Escherichia coli RecX Proteins Are Able to Replace Each Other in vivo and in vitro].

A plasmid carrying the Deinococcus radiodurans recXgene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans. RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.

[Enzymatic control of homologous recombination in Escherichia coli cells and hyper-recombination].

The RecA protein is a major enzyme of homologous recombination in bacterial cell. Forming a right-handed helical filament on ssDNA, it provides a homology search between two DNA molecules and homologous strand exchange. The RecA protein not only defends the cell from exposure to ionizing radiation and UV-irradiation, but also ensures the recombination process in the course of normal cell growth. A number of wild-type or mutant RecA proteins demonstrate increased recombinogenic properties in vitro and in vivo as compared with the wild-type RecA protein from Escherichia coli, which leads to hyper-recombination. The hyper-rec activity of RecA proteins during the recombination process in many depends on the filamentation dynamics on ssDNA and DNA-transferase properties. Changes in filamentation and DNA-transferase abilities of RecA protein may be the result of not only specific amino-acid substitutions, but also the functioning of the cell enzymatic apparatus, including such proteins as RecO, RecR, RecF, RecX, DinI, SSB, PsiB. To date, the function of each of these proteins is identified at the molecular level. However, the role of some of them in the cell metabolism remains to be seen. Increase in recombination in vivo is not always useful for a cell and faces various limitations. Moreover, in the bacterial cell some mechanisms are activated, that cause genomic reorganization, directed to suppress the expression of hyper-active RecA protein. The ways of hyper-active RecA protein regulation are very interesting, and they are studied in different model systems.

[Changing of filamentation dynamics of RecA protein, induced by D112R amino acid substitution or ATP to dATP replacement, results in filament steadiness TO THE RecX protein action].

It is known that RecX is a negative regulator of RecA protein. We found that the mutant RecA D112R protein exhibits increased resistance to RecX protein comparatively to wild-type RecA protein in vitro and in vivo. Using molecular modeling we showed, that amino acid located in position 112 can not approach RecX closer than 25-28 angstroms. Thus, direct contact between amino acid and RecX is impossible. RecA D112R protein more actively competes with SSB protein for the binding sites on ssDNA and, therefore, differs from the wild-type RecA protein by dynamics of filamentation on ssDNA. On the other hand, after the replacement of ATP by dATP, the wild-type RecA protein, changing the dynamics of filamentation on ssDNA, also becomes more resistant to RecX. Based on these data it is concluded that the dynamics of filamentation has a great, if not dominant role in the stability of RecA filament to RecX relative to the role of RecA-RecX protein-protein interactions discussed earlier. We also propose an improved model of regulation of RecA by RecX protein, where RecA filament elongation along ssDNA is blocked by RecX protein on the ssDNA region, located outside the filament.

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS.

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.

Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues.

The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain.

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP.

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the C-terminal subdomain of the protein. RecA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42 degrees C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecA-mut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.