Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI
| Autor: | Xuying Duan, Suming Tang, Guojie Zhao, Hua Wei, Yifu Guan |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
DNA Replication
0303 health sciences Loop-mediated isothermal amplification Substrate (chemistry) DNA Cell Biology 010402 general chemistry Cleavage (embryo) 01 natural sciences Biochemistry Catalysis 0104 chemical sciences 03 medical and health sciences chemistry.chemical_compound chemistry Recognition sequence Rolling circle replication Biophysics DNA Breaks Single-Stranded Locked nucleic acid Deoxyribonucleases Type II Site-Specific Molecular Biology Groove (engineering) 030304 developmental biology |
| Zdroj: | Biochemical Journal. 476:1483-1496 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bcj20190167 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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