期刊
NUCLEIC ACIDS RESEARCH
卷 43, 期 12, 页码 5998-6008出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv547
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资金
- Life Sciences Interface DTC of the UK Engineering and Physical Sciences Research Council
- European Research Council [261227]
- European Commission Seventh Framework Program [FP7] [HEALTH-F4-2008-201418]
- UK Biotechnology and Biological Sciences Research Council [BB/H01795X/1, BB/J00054X/1]
- Biotechnology and Biological Sciences Research Council [BB/J00054X/1, BB/H01795X/1] Funding Source: researchfish
- BBSRC [BB/J00054X/1, BB/H01795X/1] Funding Source: UKRI
DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.
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