Holliday Junctions Are Formed in Concentrated Solutions of Purified Products of DNA Amplification

M. G. Yakubovskaya^1*, A. A. Neschastnova¹, Zh. V. Lipatova¹, V. I. Popenko², and G. A. Belitsky¹

¹Laboratory of Carcinogen Screening Methods, Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe Shosse 24, Moscow, 115478 Russia; fax: (7-095) 323-5822; E-mail: belitsky@space.ru

²Engelhardt Institute of Molecular Biology, ul. Vavilova 32, Moscow, 117984 Russia; fax: (7-095) 135-9804; E-mail: POPENKO@genome.eimb.relarn.ru

^* To whom correspondence should be addressed.

Received February 3, 1999; Revision received August 11, 1999

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Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a “tetramer”. Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.

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KEY WORDS: DNA--DNA interaction, {}-structure, Holliday junction, PCR, DNA electrophoresis

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