Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides

“…Next, hydrolysis of 32 P-labeled modified DNA duplexes I-A to I-F by Nt.BspD6I was studied as described earlier [ 51 ]. Nonetheless, location of the modification had an influence on the initial cleavage rate ( Table 1 ).…”

“…The internucleotide phosphates with phosphodiester bonds undergoing enzyme cleavage are shown in red. Dark red arrows indicate the positions where the presence of N6-methyl-2′-deoxyadenosine in the recognition site of substrate analog blocks the Nt.BspD6I and ss.BspD6I action [ 51 ]. The positions where introduction of the non-nucleotide AB-insertion significantly decreases DNA cleavage by Nt.BspD6I are indicated by blue arrows, where influence on initial rate of hydrolysis is minimal–by the crossed blue arrows.…”

“…Nt.BspD6I and ss.BspD6I were expressed and purified separately following the protocols described in refs. [ 51 , 53 , 54 ]. Modified oligodeoxyribonucleotides were synthesized by the standard solid-phase approach using commercially available phosphoramidites (according to the protocols provided by manufacturers), then purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized as described earlier [ 55 ].…”

“…Complex formation between a protein and a 32 P-labeled DNA duplex was carried out at 37°C during 30 min in 10 μl of a buffer consisting of 10 mM Tris-HCl (pH 7.8), 150 mM KCl, 10 mM CaCl 2 , 1 mM DTT, and 0.1 mg/ml BSA; 10 nM DNA duplex and 1–100 nM Nt.BspD6I were used. To form the functional heterodimer of R.BspD6I, a 12-fold excess of the small subunit over Nt.BspD6I was added [ 51 ]. Gel electrophoresis was performed in a 7% nondenaturing polyacrylamide gel (PAG) in TBE buffer (89 mM Tris-borate, pH 8.3, 2 mM EDTA) at 15 mA.…”

“…Crystal structures are available only for free Nt.BspD6I and ss.BspD6I (PDB codes 2ewf and 2p14, respectively) without DNA substrates [ 50 ]. Previously, we have studied functional interdependence between two R.BspD6I subunits using unmodified DNA substrates and 6-methyl-2′-deoxyadenosine containing DNA [ 51 ]. Using catalytically deficient D456A and E418A variants of Nt.BspD6I, we demonstrated that the small subunit is active only in the presence of catalytically active Nt.BspD6I [ 52 ].…”