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Biomedical subjects

S M Mirkin

Publications and source records attributed to S M Mirkin.

At least 19 recordsLinked to original sources
PubMed1995

Triplex DNA structures.

A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed between triplex forming oligonucleotides (TFO) and target sequences on duplex DNA. Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs. TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo. Numerous chemical modifications of TFO are known. In peptide nucleic acid (PNA), the sugar-phosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA forming triplex with one of DNA strands leaving the other strand displaced. Very unusual recombination or parallel triplexes, or R-DNA, have been assumed to form under RecA protein in the course of homologous recombination.

Animals↗
PubMed1994

Trapping DNA polymerases using triplex-forming oligodeoxyribonucleotides.

Triplexes (triple helices) formed within DNA templates prior to or during DNA synthesis cause DNA polymerase to terminate [Samadashwily et al., EMBO J. 13 (1993) 4975-4983]. Here, we show that triplex-forming oligodeoxyribonucleotides (oligos) efficiently trap DNA polymerases at target DNA sequences within single-stranded (ss) templates. This was observed for all studied DNA polymerases, including Sequenase and the thermophilic Taq and Vent polymerases. The termination rate depends on the fine structure of a triplex, as well as on ambient conditions such as temperature and the concentration of magnesium ions. Inhibition of DNA synthesis was observed not only when triplexes blocked the path of DNA polymerase, but also when a polymerization primer was involved in triplex formation. Escherichia coli ss-binding (SSB) protein helps DNA polymerase overcome the triplex barrier, but with an efficiency dramatically dependent on the triplex configuration. These results describe a novel method for blocking DNA replication at target homopurine-homopyrimidine sequences by means of triplex-forming oligos in direct analogy with similar results during transcription.

Base Sequence↗
PubMed1992

Intramolecular DNA triplexes: unusual sequence requirements and influence on DNA polymerization.

Homopurine-homopyrimidine mirror repeats are known to form intramolecular DNA triplexes in vitro. By probing with chemical agents specific for unusual DNA conformations, we have now demonstrated the formation of intramolecular triplexes consisting of G.G.C and T.A.T base triplets by DNA sequences that are neither homopurine-homopyrimidine nor mirror repeats. This finding significantly enlarges the number of sequences that could form DNA triplexes. The observed triplexes are stable under the conditions that are optimal for DNA polymerases in vitro. We found that triplex formation causes specific termination of DNA polymerization in vitro. This effect is detected for different DNA polymerases and may have implications for the regulation of DNA replication in vivo.

Base Sequence↗
PubMed1992

Transcriptionally driven cruciform formation in vivo.

We studied the formation of d(A-T)n cruciforms in E.coli cells by probing intracellular plasmid DNA with chloroacetaldehyde followed by fine analysis of modified DNA bases. d(A-T)16 sequences were inserted into specifically designed plasmids either upstream of a single trc promoter, or between two divergent trc promoters. We found that in both cases, induction of transcription by IPTG leads to the transition of the d(A-T)16 stretch into a cruciform state. In the case of two divergent promoters, we observed cruciform formation even without IPTG. Enhanced cruciform formation correlates with the elevation in promoter activity as defined by the opening of the promoter at the -10 to +2 positions. We conclude that transcriptionally driven negative supercoiling provokes cruciform formation in vivo.

Base Sequence↗
PubMed1991

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.

Animals↗
PubMed1990

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.

Base Composition↗
PubMed1989

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH.

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.

DNA↗
PubMed1988

Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA.

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.

DNA, Superhelical↗
PubMed1988

A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs.

When plasmid DNA duplexes carrying the regular homopurine-homopyrimidine inserts (dGA)n, (dTC)n and (dG)n, (dC)n are preincubated with homologous labeled oligo(dPy) ((dTC)n and (dC)n respectively) at acid pH, the label co-electrophoreses with the duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH greater than 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed complex appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form.

DNA↗
PubMed1987

The energetics of the B-Z transition in DNA.

The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA. The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.

Chemical Phenomena↗
PubMed1987

The ionic strength dependence of the cooperativity factor for DNA melting.

The melting temperature for the d(AT)24.d(AT)24 stretch, located inside the DNA helix and terminally, have been determined in a wide range of ionic strength values (0.01 - 1 M Na+). The cooperativity factor was calculated from the shifts in the melting temperature of the stretch due to its different boundary conditions. With the sodium concentration decreasing from 1 M to 0.01 M the cooperativity factor dropped by three orders of magnitude, its change being less marked at high than at low ionic strength.

DNA↗
PubMed1987

Structure of (dG)n.(dC)n under superhelical stress and acid pH.

We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986].

DNA↗
PubMed1987

[Transfer of "artificial transposons" constructed on the basis of insertion element IS1].

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.

Bacteriophage lambda↗
PubMed1986

Structures of homopurine-homopyrimidine tract in superhelical DNA.

For homopurine-homopyrimidine tracts in superhelical DNA, we propose a structure involving Watson-Crick and Hoogsteen paired triple helixes, hairpin loops and unstructured domains. Topologically, the whole structure is equivalent to an open region. The proposed structure is consistent with available S1 cleavage, pH and alkylation data and energetics under superhelical stress; this new structure is a much more probable candidate than the one proposed by us recently (V.I. Lyamichev, S.M. Mirkin & M.D. Frank-Kamenetskii, J. Biomole. Str. Dyns 3, 327-338, 1985).

Base Sequence↗
PubMed1985

A pH-dependent structural transition in the homopurine-homopyrimidine tract in superhelical DNA.

We have inserted the 509-bp-long fragment of sea urchin P. miliaris histone gene spacer region into plasmid pUC19. The fragment contains the 60-bp-long homopurine-homopyrimidine tract that is known to be hypersensitive to the S1 endonuclease. Using two-dimensional gel electrophoresis we have observed a sharp structural transition in the insert with increasing DNA superhelicity. As in the cases of cruciform and Z form formation, the observed transition partly relaxes the superhelical stress. In contrast with the other two well documented transitions, the observed transition strongly depends on pH. At pH7 and above the transition occurs at negative superhelicities exceeding the physiological range (- sigma greater than 0.08). For pH6 the transition occurs at -sigma = 0.055, whereas for pH4.3 it takes place at -sigma = 0.001. A comprehensive analysis of the obtained data has made it possible to define the nature of the observed transition. We conclude that under superhelical stress or/and at low pH homopurinehomopyrimidine tracts adopt a novel spatial structure called the H form.

Animals↗
PubMed1985

[Development of bacteriophage Mu in E. coli gyrBts mutant strain].

The study deals with Mu growth in cells carrying a temperature-sensitive mutation in the gene of DNA gyrase B subunit. At a nonpermissive temperature the Mu growth is shown to be blocked in the host gyrB ts mutant both on infection and on prophage induction. Mu DNA does not get integrated in the host chromosome upon the infection of mutant cells, as demonstrated by DNA-DNA hybridization experiments. In the case of prophage induction in mutant cells, as opposed to the wild type cells early mRNA synthesis is practically fully inhibited while the total RNA synthesis is three times reduced after 20 min of induction. The transcription of phage DNA associated with the changed superhelicity of DNA in the cell.

Bacteriophage mu↗