Journal
NATURE COMMUNICATIONS
Volume 4, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms3131
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Funding
- European Commission [FP7/2007-2013 HEALTH-F4-2008-201418]
- UK Biotechnology and Biological Research Council [BB/H01795X/1]
- European Research Council [261227]
- US National Institutes of Health [NIH GM-28550]
- Lindemann Trust Fellowship from the English-Speaking Union
- BBSRC [BB/H01795X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/H01795X/1] Funding Source: researchfish
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The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
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