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V A Malkov

Publications and source records attributed to V A Malkov.

12 recordsLinked to original sources
PubMed2000

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex.

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.

Base Sequence↗
PubMed1998

Dissociation kinetics of RecA protein-three-stranded DNA complexes reveals a low fidelity of RecA-assisted recognition of homology.

We determined that the incorporation of one mismatch into RecA mediated synaptic complexes between oligonucleotide single-stranded DNAs and target duplex DNAs destabilizes the complex by 0.8 to 1.9 kcal/mol. This finding supports our previous result, that RecA binding per se can significantly decrease the loss in free energy associated with mismatch incorporation even in the absence of ATP hydrolysis. We show that the specificity is mostly driven by the dissociation process. We found that the relative destabilization induced by different mismatches depends on their position. Thus, while there is a good correlation between the ranking order of mismatches at the 5' end of synaptic complexes and mismatches in heteroduplexes (D-loops), there is no correlation between the ranking order for mismatches at the 3' end and mismatches in various DNA structures. This difference between the 5' and 3' ends of synaptic complexes agrees well with the established 5' to 3' polarity of the strand exchange promoted by RecA protein. The lack of a correlation between mismatches at the 3' end of synaptic complexes and mismatches in D-loops suggests the intermediate is probably not a canonical protein-free D-loop.

DNA↗
PubMed1997

Photocross-linking of the NH2-terminal region of Taq MutS protein to the major groove of a heteroduplex DNA.

The MutS DNA mismatch repair protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25-49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Taq MutS protein.

Adenosine Triphosphatases↗
PubMed1997

RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide.

We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.

Base Composition↗
PubMed1995

Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops L1 (amino acid residues 157-164) and L2 (amino acid residues 195-209).

To function as a repair and recombination protein, RecA has to be assembled as an active filament on single-stranded DNA in the presence of ATP or its analogs. We have identified amino acids in the primary DNA binding site of RecA that interact with single-stranded DNA by photocross-linking. A nucleoprotein complex consisting of RecA protein bound to a monosubstituted oligonucleotide bearing a 5-iododeoxyuracil cross-linking moiety was irradiated with long wavelength ultraviolet radiation to effect cross-linking with RecA protein. Subsequent trypsin digestion, followed by purification and peptide sequencing, revealed the cross-linking of two independent peptides, amino acid residues 153-169 and 199-216. Met164 from loop L1 and Phe203 from loop L2 were determined to be the exact points of cross-linking. Thus, our data confirm and extend predictions about the DNA binding domain of RecA protein based on the molecular structure of RecA (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).

Amino Acid Sequence↗
PubMed1994

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.

Bacterial Proteins↗
PubMed1993

Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation.

Using biotinylated oligonucleotides and streptavidin as a marker, we have visualized, with the help of electron microscopy, the triplex formation. We used the natural homopurine-homopyrimidine sequence from human papillomavirus 16 cloned within a plasmid. Under conditions favouring the formation of pyrimidine-purine-pyrimidine triplex the corresponding pyrimidine oligonucleotide formed a complex with the insert and this complex was detected by electron microscopy. Similarly, under conditions favouring the formation of pyrimidine-purine-purine triplex the corresponding purine oligonucleotide formed a stable complex detected by electron microscopy. In both cases the complexes we observed exhibited remarkable sequence specificity. Near 80% of DNA molecules carried the streptavidin marker in the correct position and very few cases of non-specific binding were detected. We conclude that the triplex mode of recognition may provide very efficient sequence-specific markers for electron microscopy of DNA.

Base Sequence↗
PubMed1993

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.

Base Sequence↗
PubMed1993

Protonated pyrimidine-purine-purine triplex.

We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions.

Base Sequence↗
PubMed1992

Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA.

We have studied the effect of intermolecular triplexes formation on the yield of cyclobutane photodimers in DNA. DNA duplex within the pyrimidine-purine-pyrimidine triplex d(TC)nd(GA)nd(CT)n is protected from the formation of cyclobutane photodimers in the case of the stabilization of this triplex by acid pH, and in the case of supplementary stabilization by Mg2+ or Zn2+. We have studied pH-independent pyrimidine-purine-purine triplexes stabilized by bivalent cations. In such triplexes, the protection from the formation of [6-4] photodimers is observed, whereas the protection from cyclobutane dimer formation does not take place. The formation of the d(TC)nd(GA)nd(GA)n triplex leads to an inversion of the intensities of cyclobutane CT and TC photodimers. We observed a sharp decrease in photoreactivity with respect to cyclobutane dimers in the duplex tract d(C)18d(G)18 in the presence of Ba2+, Cd2+, Co2+, Mn2+, Zn2+ and Ni2+. The formation of the d(C)nd(G)nd(G)n triplex leads to 'antifootprinting', i.e. an increase in the yield of cyclobutane photodimers.

Base Sequence↗
PubMed1991

Stabilization of PyPuPu triplexes with bivalent cations.

We studied the formation of stable PyPuPu intermolecular triplexes under neutral pH in the presence of bivalent cations (Mg, Ca, Mn, Co, Ni, Cu, Zn, Cd, and Ba) with the help of the photo- and DMS footprinting assays. The cations which stabilize d(C)n.d(G)n.d(G)n and d(TC)n.d(GA)n.d(AG)n triplexes were determined. Among them, Zn++ ions stabilized both triplexes, whereas Mg++ ions stabilize CGG triplexes, but do not stabilize TC.GA.AG triplexes. We have shown that an arbitrary purine sequence forms the PyPuPu triplex in the presence of Zn++ ions, and that the purine third strand is antiparallel with respect to the purine strand within the duplex.

Base Sequence↗