Dmitry O. Zharkov

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), β-elimination (AP lyase), and δ-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine‐DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential β‐elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 Å resolution. The crosslink is derived from a Schiff base intermediate that precedes β‐elimination and is stabilized by reduction with NaBH4. Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site‐directed mutagenesis studies, we propose a catalytic mechanism for Nei.

The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.

Structural characterization of the Fpg family of DNA glycosylases

Until recently, the Fpg family was the only major group of DNA glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG.

NEIL1 excises 3′ end proximal oxidative DNA lesions resistant to cleavage by NTH1 and OGG1

Base excision repair is the major pathway for the repair of oxidative DNA damage in human cells that is initiated by a damage-specific DNA glycosylase. In human cells, the major DNA glycosylases for the excision of oxidative base damage are OGG1 and NTH1 that excise 8-oxoguanine and oxidative pyrimidines, respectively. We find that both enzymes have limited activity on DNA lesions located in the vicinity of the 3′ end of a DNA single-strand break, suggesting that other enzymes are involved in the processing of such lesions. In this study, we identify and characterize NEIL1 as a major DNA glycosylase that excises oxidative base damage located in close proximity to the 3′ end of a DNA single-strand break.

Uracil-DNA glycosylase: Structural, thermodynamic and kinetic aspects of lesion search and recognition

Uracil appears in DNA as a result of cytosine deamination and by incorporation from the dUTP pool. As potentially mutagenic and deleterious for cell regulation, uracil must be removed from DNA. The major pathway of its repair is initiated by uracil-DNA glycosylases (UNG), ubiquitously found enzymes that hydrolyze the N-glycosidic bond of deoxyuridine in DNA. This review describes the current understanding of the mechanism of uracil search and recognition by UNG. The structure of UNG proteins from several species has been solved, revealing a specific uracil-binding pocket located in a DNA-binding groove. DNA in the complex with UNG is highly distorted to allow the extrahelical recognition of uracil. Thermodynamic studies suggest that UNG binds with appreciable affinity to any DNA, mainly due to the interactions with the charged backbone. The increase in the affinity for damaged DNA is insufficient to account for the exquisite specificity of UNG for uracil. This specificity is likely to result from multistep lesion recognition process, in which normal bases are rejected at one or several pre-excision stages of enzyme-substrate complex isomerization, and only uracil can proceed to enter the active site in a catalytically competent conformation. Search for the lesion by UNG involves random sliding along DNA alternating with dissociation-association events and partial eversion of undamaged bases for initial sampling.

Inactivation of mammalian 8-oxoguanine-DNA glycosylase by cadmium(II): implications for cadmium genotoxicity.

Cadmium(II) is a toxic, mutagenic and carcinogenic metal (IARC Class 1 human carcinogen). It causes damage to eukaryotic cells both in acute and chronic modes of exposure via multiple biochemical mechanisms. In particular, Cd diminishes the capacity of cells to repair oxidative DNA damage. Oxidative DNA lesions are important precursors to mutations and ultimately may lead to neoplastic transformation of human cells. We investigated interactions of Cd with murine Ogg1 (mOgg1), an enzyme that removes 8-oxoguanine (8-oxoG), an abundant oxidative lesion, from DNA. Cd(2+) and Zn(2+), but not other divalent cations tested, suppressed mOgg1-catalyzed reactions. The apparent inhibition by Cd consisted of at least two independent processes: irreversible, DNA-independent first-order inactivation of mOgg1 and DNA-dependent inhibition. Irreversibly inactivated mOgg1 has nearly normal affinity for damaged DNA and a normal catalytic rate constant but is defective in formation of the covalent reaction intermediate. When both modes of inhibition are in effect, the catalytic rate constant is dramatically lowered, while affinity to damaged DNA is decreased moderately. Potential sites for Cd binding in mOgg1 and mOgg1-DNA complex are identified. Inactivation of Ogg1 may play a role in the mutagenic and carcinogenic action of Cd.

Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower β-elimination reaction at the 3′-side of the resulting abasic site. Many features of OGG1 mechanism, including its low β-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of β-elimination rather than pre-excision dynamics of DNA and the enzyme.

Substrate specificity and excision kinetics of natural polymorphic variants and phosphomimetic mutants of human 8‐oxoguanine‐DNA glycosylase

Human 8‐oxoguanine‐DNA glycosylase (OGG1) efficiently removes mutagenic 8‐oxo‐7,8‐dihydroguanine (8‐oxoGua) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine when paired with cytosine in oxidatively damaged DNA. Excision of 8‐oxoGua mispaired with adenine may lead to G→T transversions. Post‐translational modifications such as phosphorylation could affect the cellular distribution and enzymatic activity of OGG1. Mutations and polymorphisms of OGG1 may affect the enzymatic activity and have been associated with increased risk of several cancers. In this study, we used double‐stranded oligodeoxynucleotides containing 8‐oxoGua:Cyt or 8‐oxoGua:Ade pairs, as well as γ‐irradiated calf thymus DNA, to investigate the kinetics and substrate specificity of several known OGG1 polymorphic variants and phosphomimetic Ser→Glu mutants. Among the polymorphic variants, A288V and S326C displayed opposite‐base specificity similar to that of wild‐type OGG1, whereas OGG1‐D322N was 2.3‐fold more specific for the correct opposite base than the wild‐type enzyme. All phosphomimetic mutants displayed ∼ 1.5–3‐fold lower ability to remove 8‐oxoGua in both assays, whereas the substrate specificity of the phosphomimetic mutants was similar to that of the wild‐type enzyme. OGG1‐S326C efficiently excised 8‐oxoGua from oligodeoxynucleotides and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine from γ‐irradiated DNA, but excised 8‐oxoG rather inefficiently from γ‐irradiated DNA. Otherwise, kcat values for 8‐oxoGua excision obtained from both types of experiments were similar for all OGG1 variants studied. It is known that the human AP endonuclease APEX1 can stimulate OGG1 activity by increasing its turnover rate. However, when wild‐type OGG1 was replaced by one of the phosphomimetic mutants, very little stimulation of 8‐oxoGua removal was observed in the presence of APEX1.