NDLI: Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases*

Content Provider	PubMed Central
Author	Datta, Kausiki Johnson, Neil P. Licata, Vince J. Hippel, Peter H. Von
Abstract	In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.
Related Links	http://dx.doi.org/10.1074/jbc.m109.007641
Ending Page	17193
Page Count	14
Starting Page	17180
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	25
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-06-19
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

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