Cellular DNA continually undergoes oxidative attack by reactive oxygen species. The attack leads to for- mation of AP-sites and single-strand breaks generating 3′-phosphate and 3′-phosphoglycolate ends. These are dangerous and mutagenic DNA lesions as they block DNA synthesis by DNA polymerase [1-3]. In mammalian cells, about 10,000 heterocyclic bases are known to be removed from DNA per day [4]. Non-coding AP-sites and modified 3′-ends of DNA are genotoxic, and there- fore their repair is essential for genome stability [5]. The base excision repair (BER) pathway is known to repair the majority of DNA damages [1, 6, 7]. Saccharomyces cere- visiae apurinic/apyrimidinic (AP) endonuclease 1 (Apn1) is one of the key enzymes involved in BER. The APN1 gene is located at chromosome XI followed by the RAD27 gene, the translational product of which is also involved in BER. Apn1 is a 41.4-kDa monomeric protein consisting of 367 amino acids. This enzyme possesses several cata- lytic activities: 1) AP-endonuclease activity providing DNA cleavage at the 5′-side of an ordinary or reduced AP-site; 2) 3′-phosphodiesterase activity aimed at cleav- age of 3′-blocking groups such as α,β-unsaturated alde- hydes, 4-hydroxy-2-pentenal (3′-dRP), 3′-phosphogly- colate (3′-pg), and 3′-phosphate (3′-P); 3) 3′-tyrosyl- DNA-phosphodiesterase activity leading to removal of covalently bound topoisomerase 1 (3′-Topo1); 4) endonuclease activity that provides phosphodiester bond hydrolysis from the 5′-side of oxidized DNA bases, 5,6- dihydrouracil, and formamidopyrimidines (FapyA and ISSN 0006-2979, Biochemistry (Moscow), 2012, Vol. 77, No. 10, pp. 1162-1171. © Pleiades Publishing, Ltd., 2012. Published in Russian in Biokhimiya, 2012, Vol. 77, No. 10, pp. 1401-1412. 1162 Abbreviations: Ape1, human apurinic/apyrimidinic endonucle- ase; Apn1, apurinic/apyrimidinic endonuclease from Saccha- romyces cerevisiae; AP-site, abasic site; 2-aPu, 2-aminopurine; BER, base excision repair; F, synthetic analog of AP-site ((3- hydroxytetrahydrofuran-2-yl)methyl phosphate); PyrC, pyrro- locytosine (3-[β-D-2-ribofuranosyl]-6-methylpyrrolo[2,3- d]pyrimidin-2(3H)-one). * To whom correspondence should be addressed. Kinetic Mechanism of the Interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA Substrates E. S. Dyakonova 1,2 , V. V. Koval 1,2 , A. A. Ishchenko 3 , M. K. Saparbaev 3 , R. Kaptein 2,4 , and O. S. Fedorova 1,2 * 1 Institute 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 2 Novosibirsk State University, Pirogov St. 2, 630090 Novosibirsk, Russia 3 Groupe “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 4 Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, The Netherlands; fax: 31 (0) 30-2537623; E-mail: r.kaptein@uu.nl Received March 26, 2012 Revision received May 23, 2012 Abstract—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 hete- rocyclic 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. DOI: 10.1134/S0006297912100082 Key words: Apn1, base excision repair (BER), stopped-flow method, 2-aminopurine, pyrrolocytosine