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Originally published In Press as doi:10.1074/jbc.M704341200 on August 26, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30577-30585, October 19, 2007
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Pre-steady-state Kinetic Characterization of the AP Endonuclease Activity of Human AP Endonuclease 1*Formula

Robyn L. Maher and Linda B. Bloom1

From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245

Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ' to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ' to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s–1. The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s–1. Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ' to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process.


Received for publication, May 25, 2007 , and in revised form, August 24, 2007.

* This work was supported by National Institutes of Health GM055596 (to L. B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

1 To whom correspondence should be addressed. Tel.: 352-392-8708; Fax: 352-392-6511; E-mail: lbloom{at}ufl.edu.


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S. Adhikari, A. Uren, and R. Roy
Dipole-Dipole Interaction Stabilizes the Transition State of Apurinic/Apyrimidinic Endonuclease Abasic Site Interaction
J. Biol. Chem., January 18, 2008; 283(3): 1334 - 1339.
[Abstract] [Full Text] [PDF]




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