Duplex-specific nuclease: isolation, characterization and applications
Shagin D, Bogdanova E, Zhulidov P, Wagner L, Shcheglov A.
Collaborations: Marine Biochemistry Laboratory, Pacific Institute of Bioorganic Chemistry,
Far East Branch of the Russian Academy of Sciences
Nucleases are important analytical enzymes used widely in biomedical and biotechnological applications. Their application depends on the specificity and mode of action of a particular enzyme. Nucleases with novel and unusual properties may facilitate the development of advanced technologies in many areas of biotechnology and biomedicine.
Using degenerative primers corresponding to the most conserved amino acid sequences in DNA/RNA non-specific nuclease family and Step-Out RACE technology, we isolated a cDNA comprising full coding sequence of a novel nuclease from Kamchatka crab (Paralithodes camtschaticu). The predicted crab protein shows 64% identity at the amino acid level with shrimp nuclease from Penaeus japonicus (Chou and Liao, 1990; Wang et al., 2000). Analysis of the crab sequence by SMART software (Schultz et al., 2000) disclosed that the protein comprises a 27 amino acid signal peptide and a DNA/RNA non-specific endonuclease domain (NUC domain).
The mature crab protein was expressed in E. coli. Although the recombinant protein was not active, it was used in rabbit immunization for polyclonal antibody generation. Nuclease from the kamchatka crab hepatopancreas was purified by acetone precipitation, followed by column chromatography. Fractions from the purification steps were tested for both DNase activity and antibody binding. Purified crab nuclease was present as a single band of about 42 kDa on SDS/PAGE.
Similar to other members of the DNA/RNA non-specific nuclease family, crab nuclease acquired its enzymatic activity in the presence of Mg2+ ions (7 mM required for optimal activity) and was inhibited by EDTA. The DNase activity of the purified kamchatka crab nuclease was about 18000 Kunitz units/mg protein, as determined with a modified Kunitz assay. The pH and temperature optima for activity were 7-8 and 55-65OC, respectively. The nuclease was stable at a pH of greater than 6, and temperatures below 75OC.
Surprisingly, crab nuclease exhibited strong cleavage preference for ds DNA substrates and little activity against ss DNA irrespective of the DNA length. Moreover, although the kamchatka crab nuclease was classified as part of a DNA/RNA non-specific nuclease family, no significant cleavage activity on RNA substrates was observed. However, the nuclease effectively cleaved DNA molecules in DNA-RNA hybrid duplexes. Based on these characteristics, we suggested the designation 'Duplex-Specific Nuclease' (DSN) for this enzyme. Analysis of DSN action on synthetic oligonucleotide substrates revealed that the enzyme discriminates between perfectly matched short DNA-DNA duplexes (8-12 bp) and duplexes of the same length with at least one mismatch.
A number of specific features of DSN make it ideal for removing ds-DNA from complex mixtures of nucleic acids. DSN thermal stability allows to perform ds-DNA degradation at high temperatures, thereby avoiding loss of ss DNA fraction due to the formation of secondary structures and non-specific hybridization involving adapter sequences. Using DSN we developed the novel high effective method to prepare normalized cDNA designated "DSN-normalization". Our technique efficiently normalizes cDNA samples enriched with full-length sequences, does not include laborious physical separation procedures and requires minimum hands-on time.
The ability of DSN to distinguish between variations of one nucleotide in short DNA duplexes allowed the development of a novel efficient method of SNP detection, designated the DSNP (DSN Preference) assay.