Document Detail

Dynamics of site switching in DNA polymerase.
MedLine Citation:
PMID:  23409810     Owner:  NLM     Status:  MEDLINE    
DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3' end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3'-5' exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.
Rajan Lamichhane; Svitlana Y Berezhna; Joshua P Gill; Edwin Van der Schans; David P Millar
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-03-13
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  135     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-27     Completed Date:  2013-09-09     Revised Date:  2014-03-28    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4735-42     Citation Subset:  IM    
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MeSH Terms
Base Sequence
DNA Polymerase I / chemistry,  metabolism*
DNA, Bacterial / metabolism*
Escherichia coli / chemistry,  enzymology*,  metabolism
Fluorescence Resonance Energy Transfer*
Geobacillus stearothermophilus / chemistry,  enzymology
Models, Molecular
Grant Support
Reg. No./Substance:
0/DNA, Bacterial; EC 2.7.7.-/DNA Polymerase I

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