[Complementary oligonucleotide invasion into (CA/TG)31-repetitive region of linear and circular DNA duplexes].

(CA/TG)n-repeats belong to microsatellite DNA and are the most widespread among dinucleotide repeats in mammalian genomes, occupying 0.25% of the genome. These repeats are known to be recombination "hot spots", however the molecular mechanisms of this effect are not known. We proposed that the high frequency of recombination in the repetitive regions may be due to duplex conformational characteristics resulting from a special geometry of base-stacking contacts, which permits the initiation of an invasion process of single-stranded DNA into the duplex homologous region. Here we show for the first time a DNA-DNA interaction of oligonucleotides d(CA)10 and d(TG)10 with linear and circular duplexes containing (CA/TG)31-repeats, upon incubation at 37 degrees C in the absence of proteins. Using radioactively labeled oligonucleotides, we showed that duplex-oligonucleotide interaction intensities depend on their molar ratio at a duplex concentration 30 nM. Decreasing the duplex concentration to 3 nM did not influence the intensity of oligonucleotide invasion. It was shown that more than 1%, but much less than 10% of the duplexes participate in the interaction with oligonucleotides, assuming that one molecule of the duplex interacts with one molecule of the oligonucleotide. Analysis of the kinetics of the process reveals invasion of d(CA)10 at the first minute of its incubation with the duplex, while d(TG)10 interacts with the duplex at an even higher rate. We discuss the role of DNA conformation plasticity of (CA/TG)n-repeats in the phenomenon observed, as well as its biological significance, in particular the role of CA-microsatellites in the initiation of homologous recombination.

[Chemical cleavage of DNA with single base mismatches for detection of mutations of unknown localization].

The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.

[Specific oligonucleotide invasion into the end of DNA duplex].

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.

[The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments]. / Rol' kontsevykh uchastkov dupleksov v spontannom vzaimodeistvii gomologichnykh lineinykh fragmentov DNK.

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.

[Restriction polymorphism of the proto-oncogene c-Ha-ras-1 in patients with multiple primary malignant neoplasms and non-small-cell lung cancer]. / Restriktsionnyi polimorfizm protoonkogena c-Ha-ras-1 u patsientov s pervichno-mnozhestvennymi zlokachestvennymi novoobrazovaniiami i nemelkokletochnkym rakom legkogo.

Restriction fragment length polymorphism in the human c-Ha-ras-1 locus, associated with a minisatellite sequence, was examined in 45 multiple primary cancer (MPC) patients, 56 patients with squamous cell lung cancer (SCLC), 21 patients with lung adenocarcinoma (LAC), and 53 individuals having no oncopathology. Southern analysis of cellular DNA revealed the presence of 4 common alleles (with collective allele frequency close to 94% in the control group) and a set of rare alleles. Allele a3, (2.1 kb in size under MspI/HpaII digestion) was shown to be more frequent in the MPC than in the control group. The same tendency was observed in the patients with highly differentiated cell lung cancer. An increased frequency of the a4 allele (2.5 kb under MspI/HpaII digestion) was observed in the patients with adenocarcinomas as well as in the patients with metastases and low levels of tumor tissue differentiation. The elevated frequencies of a3 in the MPC group and of a4 in the LAC patients did not correlate with increased risk of the cancers mentioned above but was associated with type of tumor progression. Previously, it was reported that the mini-satellite sequence within the c-Ha-ras-1 locus possesses enhancer activity. Our data indirectly confirm the hypothesis that the efficiency of minisatellite modulator activity is associated with fragment size.

[Microsomal monooxygenases of the liver in mice with transplanted tumors]. / Mikrosomnye monooksigenazy pecheni u myshei s perevivaemymi opukholiami.

Hepatoma 22a and Ehrlich's tumor growth were shown to be accompanied by decrease in cytochrome P-450 level in liver of noninbred and C3HA mice, these changes being more pronounced as compared to solid neoplasms. Benzo(a)pyrene-hydroxylase and amidopyrine-N-demethylase activity varied with tumor pattern. It was not changed in cases of hepatoma 22a but decreased in mice bearing Ehrlich's tumor, particularly, in those with the ascitic form. The inhibition analysis using metyrapone and 7,8-benzoflavone identified phenobarbital and methylcholanthrene forms of benzo(a)pyrene-hydroxylase in murine liver; isoform profile was not significantly affected by tumor. Liver microsomal monooxygenases of tumor-bearing mice retained inducibility by 3-methylcholanthrene.