Parker, J. B. et al. Enzymatic capture of an extrahelical thymine in the search for uracil in DNA. Nature 449, 433-437

Department of Pharmacology and Molecular Sciences, Johns Hopkins Medical School, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Nature (Impact Factor: 41.46). 10/2007; 449(7161):433-7. DOI: 10.1038/nature06131
Source: PubMed


The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

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    • "Experimental and theoretical methods have both been used to study the base flipping conformational change. The imino proton exchange with solvent during the base flipping can be measured with NMR, and is a common technique for evaluating the transition experimentally [7]. These experiments yield base opening rates as well as the equilibrium constant (K flip = k op /k clsd ) between flippedin and flipped-out state. "
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    ABSTRACT: Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1, 2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy differ-ence of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solvent interactions in implicit solvent models. We calculated electrostatic interaction energies between explicit water molecules and the base targeted for flipping. These interaction energies were calculated over the base flipping reaction coordinate to illustrate the sta-bilizing effect of the explicit water molecules on the flipped-out state. It is known that nucleic base pair hydrogen bonds also influenced the free energy of flipping since these favorable interactions must be broken in order for a base to flip-out of the helix. The Watson-Crick base pair hydrogen bond fractions were calculated over the umbrella sampling simulation windows in order to determine the effect of base pair interactions on the base flipping free energy. It is shown that interaction energies between the flip-ping base and explicit water molecules are responsible for the lower base flipping free energy difference in the explicit solvent PMF profiles.
    Preview · Article · Jan 2013 · Communications in Computational Physics
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    • "However, recent work has shown that MutM may promote lesion extrusion by inducing a bend in the DNA (Qi et al., 2009). Unlike UNG, which passively captures spontaneously extruded lesions (Parker et al., 2007), hOgg1 can dramatically bend DNA like MutM, even in the absence of a lesion, as has been directly visualized by a single-molecule imaging method (Chen et al., 2002). For hOgg1, the enzyme binding energy may strain the DNA and assist in base flipping (Friedman and Stivers, 2010); however , there is a lack of direct evidence for this mechanism. "
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    ABSTRACT: 7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative DNA lesions. 8-oxoguanine DNA glycosylases (Oggs) detect and excise 8-oxoG through a multiple-step process. To better understand the basis for estranged base recognition, we have solved the crystal structures of MBOgg1, the 8-oxoguanine DNA glycosylase of Thermoanaerobacter tengcongensis, in complex with DNA containing a tetrahydrofuranyl site (THF, a stable abasic site analogue) paired with an estranged cytosine (MBOgg1/DNA(THF:C)) or thymine (MBOgg1/DNA(THF:T)). Different states of THF (extrahelical or intrahelical) are observed in the two complexes of the ASU of MBOgg1/DNA(THF:C) structure. Analyses of their different interaction modes reveal that variable contacts on the 5' region flanking the THF abasic site are correlated with the states of the THF. Comparison of MBOgg1/DNA(THF:T) with MBOgg1/DNA(THF:C) indicates that the non-preferred estranged T may affect MBOgg1's contacts with the 5' flank of the lesion strand. Furthermore, we identified a region in MBOgg1 that is rich in positive charges and interacts with the 5' region flanking the lesion. This region is conserved only in non-eukaryotic Oggs, and additional mutagenesis and biochemical assays reveal that it may contribute to the distinct estranged base specificities between eukaryotic and non-eukaryotic Oggs.
    Full-text · Article · Dec 2012 · Journal of Structural Biology
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    • "Once the N1–C1′ glycosidic bond is cleaved, the base is further rotated (Figure 5f) and moved ∼4.6 Å to the position in the post-reactive complex where the O2 interacts with Ser271 (Figure 5g). The corresponding position of Ser271 is an asparagine in eMUG (Figure 2c) or a histidine in the UDG superfamily, including human uracil DNA glycosylase (hUNG) (41) (Supplementary Figure S5a). Since eMUG, like TDG, has activity on 5caC (Figure 2d), whereas hUNG exhibited no activity toward 5caC (6), we mutated Ser271 to histidine (S271H) in addition to alanine (S271A). "
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    ABSTRACT: The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.
    Full-text · Article · Sep 2012 · Nucleic Acids Research
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