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Nucleic Acids Research 2004 32(17):5134-5146; doi:10.1093/nar/gkh846
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Published online 30 September 2004

Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

Natalia G. Beloglazova1, Oleg O. Kirpota1, Konstantin V. Starostin1,2, Alexander A. Ishchenko1, Vitaly I. Yamkovoy2, Dmitry O. Zharkov1,2, Kenneth T. Douglas3 and Georgy A. Nevinsky1,*

1 Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Novosibirsk 630090, Russia, 2 Novosibirsk State University, 2 Pirogova Street, Novosibirsk 630090, Russia and 3 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK

* To whom correspondence should be addressed. Tel: +7 3832 35 62 26; Fax: +7 3832 33 36 77; Email: nevinsky{at}niboch.nsc.ru
Correspondence may also be addressed to Kenneth T. Douglas. Tel: +44 161 275 2371; Fax: +44 161 275 2481; Email: Ken.Douglas{at}man.ac.uk

Received June 26, 2004; Revised August 25, 2004; Accepted September 7, 2004

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude ({Delta}G° ~ –8.7 to –9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude ({Delta}G° ~ –1.1 to –1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.


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