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Nucleic Acids Res. 2012 Sep;40(16):7975-84. doi: 10.1093/nar/gks523. Epub 2012 Jun 4.

Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I.

Author information

1
Department of Chemistry, Wayne State University, Detroit, MI, USA.

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.

PMID:
22669904
PMCID:
PMC3439913
DOI:
10.1093/nar/gks523
[Indexed for MEDLINE]
Free PMC Article

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