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

Veronika E Anisimova¹^,2 , Denis V Rebrikov¹ , Dmitry A Shagin¹^,2 , Valery B Kozhemyako³ , Natalia I Menzorova³ , Dmitry B Staroverov¹ , Rustam Ziganshin¹ , Laura L Vagner¹ , Valery A Rasskazov³ , Sergey A Lukyanov¹ and Alex S Shcheglov¹

¹M.M. Shemiakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia

²Evrogen JSC, Miklukho-Maklaya 16/10, 117871 Moscow, Russia

³Pacific Institute of Bioorganic Chemistry, RAS Far East Division, 690022 Vladivostok, Russia

author email corresponding author email

BMC Biochemistry 2008, 9:14doi:10.1186/1471-2091-9-14


Published:	21 May 2008

Abstract

Background

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.

Results

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.

Conclusion

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.