气候变化领域集成服务门户(CCPortal): Real-time observation of cas9 postcatalytic domain motions

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DOI	10.1073/pnas.2010650118
	Real-time observation of cas9 postcatalytic domain motions
	Wang Y.; Mallon J.; Wang H.; Singh D.; Jo M.H.; Hua B.; Bailey S.; Ha T.
发表日期	2021
ISSN	00278424
卷号	118 期号:2
英文摘要	CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3′ flap generated by NTS cleavage can be rapidly digested by a 3′ to 5′ single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3′ flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9’s conformation and suggest possible biotechnological applications. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Conformational rearrangement; CRISPR-cas9; Single molecule
语种	英语
scopus关键词	CRISPR associated endonuclease Cas9; exonuclease; magnesium ion; single stranded DNA; Article; biochemical analysis; biotechnology; catalysis; conformational rearrangement; controlled study; coordinated domain motion; cryoelectron microscopy; DNA cleavage; DNA repair; enzyme conformation; fluorescence resonance energy transfer; NTS digestion assay; priority journal; protein domain; protein structure; radio labeled cleavage assay; regulatory mechanism; single molecule assay; single molecule data analysis
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/181096
作者单位	Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States; Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, United States; HHMI, Johns Hopkins University, Baltimore, MD 21205, United States; Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, United States; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, United States
推荐引用方式 GB/T 7714	Wang Y.,Mallon J.,Wang H.,et al. Real-time observation of cas9 postcatalytic domain motions[J],2021,118(2).
APA	Wang Y..,Mallon J..,Wang H..,Singh D..,Jo M.H..,...&Ha T..(2021).Real-time observation of cas9 postcatalytic domain motions.Proceedings of the National Academy of Sciences of the United States of America,118(2).
MLA	Wang Y.,et al."Real-time observation of cas9 postcatalytic domain motions".Proceedings of the National Academy of Sciences of the United States of America 118.2(2021).