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Kinetic Mechanism of the Interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA Substrates

E. S. Dyakonova1,2, V. V. Koval1,2, A. A. Ishchenko3, M. K. Saparbaev3, R. Kaptein2,4, and O. S. Fedorova1,2*

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave. 8, 630090 Novosibirsk, Russia; fax: (383) 363-5153; E-mail: fedorova@niboch.nsc.ru

2Novosibirsk State University, Pirogov St. 2, 630090 Novosibirsk, Russia

3Groupe “Reparation de l’ADN” Univ. Paris-Sud XI, UMR 8200 C.N.R.S. Institut Gustave Roussy Villejuif Cedex F-94805, France; fax: 33 (1) 42115008; E-mail: murat.saparbaev@igr.fr

4Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, The Netherlands; fax: 31 (0) 30-2537623; E-mail: r.kaptein@uu.nl

* To whom correspondence should be addressed.

Received March 26, 2012; Revision received May 23, 2012
The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306-1321) for human Ape1 revealed some differences in their interaction with DNA substrates.
KEY WORDS: Apn1, base excision repair (BER), stopped-flow method, 2-aminopurine, pyrrolocytosine

DOI: 10.1134/S0006297912100082