[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.

[Instability of plasmid DNA integrated into mammalian somatic cell genome]. / Izuchenie iavleniia nestabil'noi integratsii chuzherodnoi DNK v genom somaticheskikh kletok mlekopitaiushchikh.

The phenomenon of loosing exogenic DNA from the mammalian somatic cell genome is under investigation. It is found that foreign DNA incorporated into cell genome as a result of transfection by electrophoretion may be lost with the frequency from 1/100 up to 1/100 000 per cell division during cultivation. This effect is not dependent of the nature of cell line and vector DNA. It is actual for different cell lines: A23, human fibroblasts AG 11395, murine embryonic line F9, and for different plasmid vectors: p16, p.39, pATR4 and pcDNA3.1-Higr (WRN). Integration of pDNA into genome and the following loosing of this DNA is registered by selection markers G418 and hygromycin B resistance and gancyclovir sensibility. The presence of foreign DNA in the genome was controlled by PCR. It is found that true foreign DNA deletion from the genome takes place rather than gene expression changes. For closely linked plasmid genes deletion of both genes at once as well as loosing any one gene separately is shown. Thus, the phenomenon of selective deletion of exogenic DNA from genome has been demonstrated for different mammalian cells.

Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli): a search for RecA protein regions responsible for this activity.

In the background of weak, if any, constitutive SOS function, RecA from Pseudomonas aeruginosa (RecAPa) shows a higher frequency of recombination exchange (FRE) per DNA unit length as compared to RecA from Escherichia coli (RecAEc). To understand the molecular basis for this observation and to determine which regions of the RecAPa polypeptide are responsible for this unusual activity, we analyzed recAX chimeras between the recAEc and recAPa genes. We chose 31 previously described recombination- and repair-proficient recAX hybrids and determined their FRE calculated from linkage frequency data and constitutive SOS function expression as measured by using the lacZ gene under control of an SOS-regulated promoter. Relative to recAEc, the FRE of recAPa was 6.5 times greater; the relative alterations of FRE for recAX genes varied from approximately 0.6 to 9.0. No quantitative correlation between the FRE increase and constitutive SOS function was observed. Single ([L29M] or [I102D]), double ([G136N, V142I]), and multiple substitutions in related pairs of chimeric RecAX proteins significantly altered their relative FRE values. The residue content of three separate regions within the N-terminal and central but not the C-terminal protein domains within the RecA molecule also influenced the FRE values. Critical amino acids in these regions were located close to previously identified sequences that comprise the two surfaces for subunit interactions in the RecA polymer. We suggest that the intensity of the interactions between the subunits is a key factor in determining the FRE promoted by RecA in vivo.

Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells.

The replacement of Escherichia coli recA gene (recA[Ec]) with the Pseudomonas aeruginosa recA(Pa) gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA(Pa) protein with those of RecA(Ec) protein. Consistent with hyper-recombination activity, RecA(Pa) protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA(Pa) protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA(Ec) protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA(Pa) protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA(Pa) in comparison with RecA(Ec), RecA441 and RecA803 proteins, RecA(Pa) showed unusually high abilities: to be resistant to the displacement by SSB 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 pH7.5. 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.

Genetic characteristics of new recA mutants of Escherichia coli K-12.

To search for functionally thermosensitive (FT) recA mutations, as well as mutations with differently affect RecA protein functions, seven new recA mutations in three different regions of the RecA protein structure proposed by Story et al. [R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992] were constructed. Additionally, the recA2283 allele responsible for the FT phenotype of the recA200 mutant was sequenced. Five single mutations (recA2277, recA2278, recA2283, recA2283E, and recA2284) and one double mutation (recA2278-5) generated, respectively, the amino acid substitutions L-277-->N, G-278-->P, L-283-->P, L-283-->E, I-284-->D, and G-278-->T plus V-275-->F in the alpha-helix H-beta-strand 9 region of the C-terminal domain of the RecA protein structure. According to recombination, repair, and SOS-inducible characteristics, these six mutations fall into four phenotypic classes: (i) an FT class, with either inhibition of all three analyzed functions at 42 degrees C (recA2283), preferable inhibition at 42 degrees C of recombination and the SOS response (recA2278), or inhibition at 42 degrees C of only recombination (recA2278-5); (ii) a moderately deficient class (recA2277); (iii) a nondeficient class (recA2283E); and (iv) a mutation with a null phenotype (recA2284). The recA2223 mutation generates an L-223-->M substitution in beta-strand 6 in a central domain of the RecA structure. This FT mutation shows preferable inhibition of the SOS response at 42 degrees C. The recA2183 mutation produces a K-183-->M substitution in alpha-helix F of the same domain. The Lys-183 position in the Escherichia coli RecA protein was found among positions which are important for interfilament interaction (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992).

[Recombination properties of the modified Pseudomonas aeruginosa RecA protein]. / Rekombinatsionnye svoistva modifitsirovannogo belka RecA iz Pseudomonas aeruginosa.

Gene recA from Pseudomonas aeruginosa was cloned into pUC19 vector under lacZ promoter. The expressed protein appeared to be modified, the aminoterminal part of deduced amino acid sequence of the RecAPa protein was found elongated by a polypeptide of 10 amino acids. The modified protein named RecA*Pa completely replaces RecAEc from E. coli in vivo recombination. In vitro RecA*Pa promotes the homologous strand transfer from a short linear duplex DNA fragment (346 bp) into circular single-stranded DNA (8196 n) being 5 times more active than RecAEc. However, when the length of dsDNA increased the difference between two proteins becomes negligible. To understand the reasons, some properties of RecA*Pa and RecAEc were compared. The former was shown to be more active both in binding to ssDNA in ssDNA-dependent ATP hydrolysis. The Rec*Pa protein showed also a high affinity to dsDNA, even at a physiological pH which is known to be unfavorable for RecAEc/dsDNA binding. However, both proteins equally catalyzed the dsDNA-dependent ATP hydrolysis; we suggest that this is crucial for a full-length DNA strand transfer recombination reaction.

Functional characteristics of the recA gene from Serratia marcescens strain Sb.

The cloned recA gene from Serratia marcescens Sb was expressed and complemented defects in the UV repair, recombination, and SOS induction of an Escherichia coli host deleted for recA. Moreover, the Serratia gene, recA (Sm), supported the same frequency of recombination per unit length of DNA as did the homologous Escherichia coli gene, recA(Ec).

[Comparative analysis of the structure and function of recA genes from Serratia marcescens Sb and Escherichia coli K-12]. / Sravnitel'nyi analiz struktury i funktsii genov recA iz Serratia marcescens Sb i Escherichia coli K-12.

Nucleotide sequence of the 1276 bp fragment of Serratia marcescens DNA coding for the recASM gene has been determined. This structure was shown to contain an ORF corresponding to a protein with molecular weight of 37766 D. Comparative analysis of the regulatory part of recASM and recAEC (Escherichia coli) demonstrated identity of "-35" and "-10" boxes for these genes and similarity of the SOS box and the enhancer sequences. A comparison of the amino acids sequences of RecASM, RecAEC and RecAPA (Pseudomonas aeruginosa) proteins revealed a great conservatism in the N-terminus and in some structural patches (alpha-helices and beta-sheets) of the RecA proteins predicted by the model of Blanar et al. In contrast, a strong variability of the C-terminus (for the last 25 amino acids, in particular) was revealed. A necessity for definite amino acids composition of the carboxy-terminal end is discussed.

[Cloning and characteristics of recA gene in Pseudomonas aeruginosa]. / Klonirovanie i kharakteristika gena recA iz Pseudomonas aeruginosa.

A recA-like gene from Pseudomonas aeruginosa was cloned and identified by means of interspecific complementation of gene recA repair defect in Escherichia coli. The gene was mapped in the PvuII-HindIII Ps. aeruginosa chromosome fragment of 1.5 kbp in length. Having been recloned in pUC18 or 19 plasmids in either of possible orientations, this fragment was shown to complement three different defects of E. coli recA mutants: in repair, recombination and SOS functions.