Abstract
BackgroundNucleases, 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.ResultsUsing 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.ConclusionWe 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.
Highlights
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
Because the discovery of nucleases with unique properties has often led to the development of new molecular biology methods, the search for novel nucleases remains an important scientific endeavor
Applying the rapid amplification of cDNA ends (RACE) procedure, we amplified the 3'-terminus of Duplex-Specific Nuclease (DSN) cDNA to obtain a 600-base-pairs PCR product
Summary
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. Among these are restriction endonucleases that target specific DNA sequences, as well as DNA-RNA nonspecific endonucleases that hydrolyze nucleic acids and nucleoside monophosphates [1] These enzymes are key components of many biologically important processes, including DNA replication, repair and recombination (including transposition), and antiviral defense, apoptosis and digestion. Because of their versatility and specificity, these enzymes have revolutionized molecular biology, making possible a host of modern techniques, including molecular cloning, studies of DNA-protein interaction and determination of nucleic acid structures. These results provide the first evidence that enzymes belonging to this family of non-specific DNARNA endonucleases may target ds DNA-containing substrates
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