Main properties of DSN

Substrate specificity: DSN exhibits strong cleavage preference for ds DNA substrates. It can show minor activity against ss DNA when both DSN enzyme and substrate are present at high concentrations, however this activity is not detectable in presence of competitive ds DNA and RNA (Zhao et al., 2008). DSN does not cleave RNA, but effectively cleaves DNA in DNA-RNA hybrid duplexes (Shagin et al., 2002)

	Action of DSN on ss DNA of phage M13 and ds DNA of phage λ. Lanes 1, 2 – negative controls, incubation without nuclease. Lane 1: phage M13 DNA alone, Lane 2 – mixture containing phage M13 and λ DNA. Lanes 3; 4 – digestion of phage M13 and λ DNA mixture by DSN at 70°C for 1.5 min (lane 3) and 5 min (lane 4).

Action of DSN on ss DNA of phage M13 and ds DNA of phage λ.

Lanes 1, 2 – negative controls, incubation without nuclease. Lane 1: phage M13 DNA alone, Lane 2 – mixture containing phage M13 and λ DNA. Lanes 3; 4 – digestion of phage M13 and λ DNA mixture by DSN at 70°C for 1.5 min (lane 3) and 5 min (lane 4).

Analysis of DSN activity on synthetic oligonucleotide substrates revealed that the enzyme discriminates between perfectly matched short DNA-DNA duplexes (10-12 bp) and duplexes of the same length with at least one mismatch. It requires at least 10 bp DNA or 15 bp DNA-RNA perfect duplex for cleavage.

	DSN action on one mismatch-containing and perfectly matched DNA duplexes. Duplexes formed by 5-carboxyfluorescein (Fl)-5'-gccctatagt-3'-TAMRA signal probe and complementary targets (5’-actcactataCggcgaat-3’ and 5’-actcactatagggcgaat-3’) were incubated with DSN at 35°C for 15 min. Emission spectra were obtained on the spectrofluorimeter, with excitation at 480 nm. Dotted line – substrate fluorescence in the absence of enzyme; firm line – substrate fluorescence after incubation with DSN.

DSN action on one mismatch-containing and perfectly matched DNA duplexes.

Duplexes formed by 5-carboxyfluorescein (Fl)-5'-gccctatagt-3'-TAMRA signal probe and complementary targets (5’-actcactataCggcgaat-3’ and 5’-actcactatagggcgaat-3’) were incubated with DSN at 35°C for 15 min. Emission spectra were obtained on the spectrofluorimeter, with excitation at 480 nm. Dotted line – substrate fluorescence in the absence of enzyme; firm line – substrate fluorescence after incubation with DSN.