Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

<p>Abstract</p> <p>Background</p> <p>Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.</p> <p>Results</p> <p>Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.</p> <p>Conclusion</p> <p>We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.</p

АКАДЕМИК Н.А. ЛОГАЧЕВ И ЕГО НАУЧНАЯ ШКОЛА: ВКЛАД В ИЗУЧЕНИЕ КАЙНОЗОЙСКОГО КОНТИНЕНТАЛЬНОГО РИФТОГЕНЕЗА

N.A. Florensov and N.A. Logatchev pioneered development of fundamental concepts of the structure and evolution of the Baikal system of rift basins. At the turn to the 21st century, in view of the wide availability of scientific research data on the Cenozoic continental rift zones located in Eurasia, Africa and North America, and taking into account the application of new research methods and options to process and analyze huge amounts of geological and geophysical data, a priority was comprehensive modeling of rifting from its origin to the current period of time. This scientific challenge was addressed by the research team under the leadership of N.A. Logachev.Фундаментальные представления о строении и развитии Байкальской системы рифтовых впадин были заложены в трудах Н.А. Флоренсова и Н.А. Логачева. Высокая степень изученности кайнозойских континентальных рифтовых зон Евразии, Африки и Северной Америки, а также новые методики и возможности обработки и анализа больших массивов геологической и геофизической информации выдвинули на рубеже XX и XXI столетий в качестве приоритетной задачи создание комплексной модели развития рифтогенеза с его зарождения до современности. Решение поставленной задачи осуществлялось в рамках работ научной школы под руководством Н.А. Логачева

A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed “duplex-specific nuclease preference” (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA. [Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank/EMBL/Date Bank under accession nos. AF520591. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N.K. Yankovsky, A.V. Polyakov, and G.N. Rudenskaya.

ACADEMICIAN N.A. LOGATCHEV AND HIS SCIENTIFIC SCHOOL: CONTRUBITION TO STUDIES OF THE CENOZOIC CONTINENTAL RIFTING

N.A. Florensov and N.A. Logatchev pioneered development of fundamental concepts of the structure and evolution of the Baikal system of rift basins. At the turn to the 21st century, in view of the wide availability of scientific research data on the Cenozoic continental rift zones located in Eurasia, Africa and North America, and taking into account the application of new research methods and options to process and analyze huge amounts of geological and geophysical data, a priority was comprehensive modeling of rifting from its origin to the current period of time. This scientific challenge was addressed by the research team under the leadership of N.A. Logachev

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel <em>Crenomytilus grayanus</em>

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties