Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Nucleic Acids Research (Impact Factor: 9.11). 05/2009; 37(11):3493-500. DOI: 10.1093/nar/gkp161
Source: PubMed


The DNA repair enzyme human uracil DNA glycosylase (UNG) scans short stretches of genomic DNA and captures rare uracil bases as they transiently emerge from the DNA duplex via spontaneous base pair breathing motions. The process of DNA scanning requires that the enzyme transiently loosen its grip on DNA to allow stochastic movement along the DNA contour, while engaging extrahelical bases requires motions on a more rapid timescale. Here, we use NMR dynamic measurements to show that free UNG has no intrinsic dynamic properties in the millisecond to microsecond and subnanosecond time regimes, and that the act of binding to nontarget DNA reshapes the dynamic landscape to allow productive millisecond motions for scanning and damage recognition. These results suggest that DNA structure and the spontaneous dynamics of base pairs may drive the evolution of a protein sequence that is tuned to respond to this dynamic regime.

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    • "This allows potential substrates to be identified without fully inserting every base into the active site pocket (Fig. 2) (Banerjee et al. 2005; Parker et al. 2007). Consistently, NMR studies on human UNG showed the glycosylase to undergo a conformational change upon DNA binding, allowing for oscillation between an open form, loosely interacting with the DNA in an unspecific manner, and a closed form engaged in base examination without fully rotating the base out of the helix (Fig. 2) (Friedman et al. 2009). Base pre-scanning is likely to be facilitated by DNA breathing, considering that many base lesions affect base pairing dynamics to some extent and are therefore likely to enhance local DNA melting. "
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    ABSTRACT: The base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy. All glycosylases share a common mode of action for damage recognition; they flip bases out of the DNA helix into a selective active site pocket, the architecture of which permits a sensitive detection of even minor base irregularities. Within the past few years, it has become clear that nature has exploited this ability to read the chemical structure of DNA bases for purposes other than canonical DNA repair. DNA glycosylases have been brought into context with molecular processes relating to innate and adaptive immunity as well as to the control of DNA methylation and epigenetic stability. Here, we summarize the key structural and mechanistic features of DNA glycosylases with a special focus on the mammalian enzymes, and then review the evidence for the newly emerging biological functions beyond the protection of genome integrity.
    Chromosoma 11/2011; 121(1):1-20. DOI:10.1007/s00412-011-0347-4 · 4.60 Impact Factor
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    • "In summary, a series of discrimination events induce the specific helical extrusion and entry of oxoG into the enzyme's activie site (34). The proposed direct recognition of the intrahelical oxoG by OGG and MutM contrasts with the suggested mechanism by which uracil DNA-glycosylase (UNG) finds and repairs deoxyuridine (dU) in DNA (38–41). In the case of dU, the experimental data are consistent with UNG passively finding and trapping extrahelical dU residues rather than facilitating the selective extrusion of the cognate dU from the DNA stack base. "
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    ABSTRACT: The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.
    Nucleic Acids Research 05/2011; 39(15):6789-801. DOI:10.1093/nar/gkr275 · 9.11 Impact Factor
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    ABSTRACT: 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.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/2009; 685(1-2):11-20. DOI:10.1016/j.mrfmmm.2009.10.017 · 3.68 Impact Factor
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