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Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI
Hua Wei1,2, Suming Tang1, Xuying Duan1
1Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110001, China.
The Biochemical Journal
|May 9, 2019
Summary
This study reveals how the nicking endonuclease Nt.BstNBI binds DNA using rolling circle amplification. Findings pinpoint specific DNA grooves and positions Nt.BstNBI interacts with during nicking.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Nicking endonucleases (NEs) are crucial for isothermal amplification techniques.
- Limited crystal structures hinder detailed catalytic mechanism studies of many NEs, including Nt.BstNBI.
- Understanding Nt.BstNBI's mechanism is essential for optimizing its applications.
Purpose of the Study:
- To elucidate the catalytic mechanism of the nicking endonuclease Nt.BstNBI.
- To identify the specific DNA binding positions and regions utilized by Nt.BstNBI.
- To demonstrate the utility of rolling circle amplification (RCA) for studying nuclease-DNA interactions.
Main Methods:
- Utilized rolling circle amplification (RCA) assay as the primary analytical tool.
- Employed locked nucleic acid (LNA) modification for DNA substrate analysis.
- Designed DNA duplex length and mismatch variations to probe substrate interactions.
Main Results:
- Identified potential binding sites for Nt.BstNBI within the major and minor grooves of the DNA substrate.
- Determined that Nt.BstNBI recognizes six adjacent positions in the major groove (G1rt, A2rt, G3rt, A2rb, C3rb, T4rb).
- Found that Nt.BstNBI holds three positions in the minor groove (N3ct, N4ct, N7cb) for DNA cleavage.
- Demonstrated RCA's efficiency and high-throughput capability for studying nuclease-macromolecular interactions.
Conclusions:
- Nt.BstNBI likely recognizes specific sequences in both the major and minor grooves of DNA for nicking.
- RCA is a powerful and accessible method for investigating the mechanisms of certain nucleases.
- This research provides novel insights into Nt.BstNBI's DNA binding and catalytic mechanism.
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