Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I.
Author(s)
Markiewicz, RP
Vrtis, KB
Rueda, D
Romano, LJ
Type
Journal Article
Abstract
The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus.
Date Issued
2012-09-01
Start Page
7975
End Page
7984
Journal / Book Title
Nucleic Acids Res
Volume
40
Issue
16
Copyright Statement
© The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
License URL
Description
07.08.14 KB. Ok to add published version to spiral, OA paper
Identifier
http://www.ncbi.nlm.nih.gov/pubmed/22669904
gks523
Coverage Spatial
England