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E N Zaitsev

Publications and source records attributed to E N Zaitsev.

11 recordsLinked to original sources
PubMed2000

A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange.

Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this "inverse" reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein-dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication.

Base Sequence↗
PubMed1999

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein.

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Adenosine Triphosphate↗
PubMed1999

The DNA binding properties of Saccharomyces cerevisiae Rad51 protein.

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.

Adenosine Triphosphate↗
PubMed1999

Enhanced monomer-monomer interactions can suppress the recombination deficiency of the recA142 allele.

The RecA142 protein, in which valine is substituted for isoleucine-225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo function of RecA142 protein, suggesting that recombination function can be restored by a slight change in protein structure elicited by protonation. Indeed, we identified an intragenic suppressor of the recombination deficiency of the recA142 allele. This suppressor mutation is a substitution of leucine for glutamine at position 124. Based on the three-dimensional structure, the Q-124L substitution is predicted to make a new monomer-monomer contact with residue phenylalanine-21 of the adjacent RecA monomer. The Q-124L mutation is not allele specific, because it also suppresses the recombination deficiency of a recA deletion (Delta9), lacking nine amino acids at the amino-terminus, presumably by reinforcing the monomer-monomer interactions that are attenuated by the Delta9 deletion. Expression of RecA(Q-124L) protein is toxic to Escherichia coli, presumably because of enhanced affinity for DNA. We speculate as to how enhanced monomer-monomer interactions and acidic pH conditions can restore the recombination activity of some defective recA alleles.

Acids↗
PubMed1998

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.

Adenosine Triphosphate↗
PubMed1996

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).

Alleles↗
PubMed1991

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).

Cloning, Molecular↗
PubMed1991

Regenerative life-support system development problems for the Mars mission.

An analysis of expediency of using physicochemical and biotechnological complexes of life-support systems has been performed with reference to the most likely trends of manned cosmonautics development for the nearest decades--orbital stations in the Earth orbit, Moon base, and Mars mission. The analysis results have shown that the physicochemical complex is much more advantageous than the biotechnological one for all cases considered. This conclusion is based on significant difference in power utilization factor: 70-90% for the physicochemical complex and 5-10% for the biotechnological one as far as the photosynthesis efficiency is concerned. The selection of the physicochemical complex systems is considered; particular attention is payed to operating reliability and performance.

Biotechnology↗
PubMed1984

Native supercoiling of DNA: the effects of DNA gyrase and omega protein in E. coli.

This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrBts) and a deletion of the top gene encoding the omega protein upon the superhelical density of the pAO3 plasmid in E. coli cells. The alteration of the DNA gyrase B subunit is shown to lead to a partial relaxation of DNA. On the other hand, the lack of omega protein due to the top gene deletion leads to an abnormally high degree of DNA supercoiling. In a double gyrBts delta top mutant the DNA supercoiling is greater than native at the permissive temperature, while under nonpermissive conditions a partial relaxation is observed. However, the pattern of DNA relaxation in the latter case is quite different from that in a single gyrBts mutant. The conclusion is that the native supercoiling of DNA in the cell is maintained through the counter-activities of DNA gyrase and the omega protein.

DNA Topoisomerases, Type I↗
PubMedDate not supplied

Long-duration space mission regenerative life support.

The paper deals with the construction of physical/chemical life support systems of orbiting space station Mir and the Russian segment of the international space station (ISS). Based on experience gained in development and long-term operation of systems for water recovery and air revitalization balance and energy/mass characteristics of promising life support systems (LSS) are analyzed. Physical/chemical life support systems with regenerative systems updated as a result of the operation on the ISS may be used at an initial phase of manned interplanetary missions.

Air Conditioning↗