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

Screening substrate binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI.
Biochem J. 2019 May 07;:
Authors: Wei H, Tang S, Duan X, Guan Y, Zhao G
Abstract
Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplifications. 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 (LNA) 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, T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, 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.
PMID: 31064800 [PubMed - as supplied by publisher]

Source: The Biochemical Journal - May 6, 2019 Category: Biochemistry Authors: Wei H, Tang S, Duan X, Guan Y, Zhao G Tags: Biochem J Source Type: research