Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI

Abstract

Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

“…We should mention that the data presented in ref. [12] are in line with our earlier results on Nt.BspD6I [10,11]. The structural organisation of Nt.BspD6I [8] and results from ref.…”

“…The structural organisation of Nt.BspD6I [8] and results from ref. [12] have further confirmed the existence of separate recognition and cleavage domains in Nt.BstNBI from a biochemical point of view and indicate that the catalytic domain of this NE has great potential for the construction of chimeric endonucleases containing recognition domains with other sequence specificity.…”

“…Information about the mechanism of functioning of other NEs in the literature is also lacking. The only paper was published in 2019, where a model of substrate interaction of Nt.BstNBI (isoschizomer of Nt.BspD6I) was suggested using locked-nucleic-acid substrate analogues and the rolling circle amplification technique [12]. We should mention that the data presented in ref.…”