Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

Abstract

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

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

“…7) observed after applying 1 μM of Nt.BspD6I. It is previously shown, that Nt.BspD6I is able to oligomerize at concentrations higher than 0.5 μM [31]. Using the frequency change of 200 Hz and molecular weight of Nt.BspD6I (70.8 kDa) the surface concentration of NE can be estimated according to Eq.…”

“…However, the fact of Nt.BspD6I complex formation with product of its DNA hydrolysis was demonstrated by both approaches. It was previously shown that Nt.BspD6I possesses the ability of nonspecific DNA binding [31]. To assess the extent of such non-specific binding with DNA, we analyzed the hydrolysis of duplex IV that contained fluorescent label TAMRA on the 3′-end of its top strand in the presence of different competitors: canonical duplex I-F, duplex I-G that corresponds to the product of hydrolysis by Nt.BspD6I, and 30-bp non-specific duplex III without the Nt.BspD6I recognition site (Fig.…”

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

“…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 ].…”

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

“…It has been established by an electrophoretic mobility shift assay [47] that Nt.BspD6I has high affinity for non-specific DNA duplexes. It was shown in refs.…”