Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Journal of Biological Chemistry (Impact Factor: 4.65). 04/2012; 287(30):24916-28. DOI: 10.1074/jbc.M111.316497
Source: PubMed

ABSTRACT A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.

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    ABSTRACT: 8-Oxoguanine-DNA glycosylase (OGG1) removes a pre-mutagenic lesion 8-oxoguanine (oxoGua) from DNA and then nicks the nascent abasic (AP) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of oxoGua recognition are not well comprehended. In order to understand the mechanisms of substrate recognition and processing we have constructed OGG1 mutants with the active site occluded by replacement of Cys253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants was characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild-type and mutant OGG1-substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of oxoGua and AP sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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    ABSTRACT: Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids. Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA-substrates were examined. The roles of catalytically important amino acids F45, Y203, K249, H270, F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated. The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently. General significance The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.
    Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
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    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.
    PLoS ONE 01/2014; 9(6):e98495. · 3.53 Impact Factor

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