Nucleic Acids Research Advance Access originally published online on August 30, 2007
Nucleic Acids Research 2007 35(18):6052-6062; doi:10.1093/nar/gkm587
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Nucleic Acids Research, 2007, Vol. 35, No. 18 6052-6062
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06520, USA
*To whom correspondence should be addressed. Tel: +1 203 785 4599; Fax: +1 203 785 7979; Email: William.Konigsberg{at}yale.edu
Received June 1, 2007. Revised July 13, 2007. Accepted July 17, 2007.
We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.