Thermodynamics of the multi-stage DNA lesion recognition and repair by Formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence - Stopped-flow pre-steady-state kinetics

Nikita A. Kuznetsov, Yuri N. Vorobjev, Lev N. Krasnoperov, Olga S. Fedorova

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8- oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10-30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van't Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.

Original languageEnglish (US)
Pages (from-to)7384-7392
Number of pages9
JournalNucleic Acids Research
Volume40
Issue number15
DOIs
StatePublished - Aug 2012

All Science Journal Classification (ASJC) codes

  • Genetics

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