Abstract
Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.
Highlights
DNA is continuously damaged by reactive oxygen species (ROS) generated by UV light, ionizing radiation and during metabolism [1,2,3,4,5]
HOGG1 is a bifunctional enzyme, possessing DNA glycosylase activity and AP lyase activity
In the previous studies we investigated the conformational dynamics of hOGG1 and DNA substrates during the catalytic cycles using fluorescence detection [14,20]
Summary
DNA is continuously damaged by reactive oxygen species (ROS) generated by UV light, ionizing radiation and during metabolism [1,2,3,4,5]. Among the various products of oxidative stress, 7,8-dihydro-8-oxoguanosine (oxoG) is the most commonly found. It is a pre-mutagenic DNA lesion since oxoG is able to mispair with adenine, generating G/C to T/A transversion mutations [6]. As is the case of most oxidized bases, oxoG is primarily removed in the base excision repair (BER) pathway. This pathway is initiated by the recognition of the modified bases by specific DNA glycosylases. HOGG1 is a bifunctional enzyme, possessing DNA glycosylase activity (hydrolysis of the N-glycosidic bond of the damaged nucleotide resulting in formation of the abasic product, Fig. 1) and AP lyase activity (elimination of the 39phosphate, often referred to as b-elimination resulting in formation of the nicked product, Fig. 1). In contrast to several other bifunctional DNA glycosylases, the AP lyase activity of hOGG1 is much weaker than its glycosylase activity [11,12,13,14]
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