Dataset Information

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

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.

SUBMITTER: Zharkov DO

PROVIDER: S-EPMC3000906 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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

Zharkov Dmitry O DO Mechetin Grigory V GV Nevinsky Georgy A GA

Mutation research 20091110 1-2

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 fro ...[more]

PMID: 19909758

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Project description:Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.

Project description:The family 4 uracil-DNA glycosylase from the hyperthermophilic organism Archaeoglobus fulgidus (AFUDG) is responsible for the removal of uracil in DNA as the first step in the base excision repair (BER) pathway. AFUDG contains a large solvent-exposed peptide region containing an α helix and loop anchored on each end via ligation of two cysteine thiolates to a [4Fe-4S](2+) cluster. We propose that this region plays a similar role in DNA damage recognition as a smaller iron-sulfur cluster loop (FCL) motif in the structurally unrelated BER glycosylases MutY and Endonuclease III and therefore refer to this region as the "pseudo-FCL" in AFUDG. In order to evaluate the importance of this region, three positively charged residues (Arg 86, Arg 91, Lys 100) and the anchoring Cys residues (Cys 85, Cys 101) within this motif were replaced with alanine, and the effects of these replacements on uracil excision in single- and double-stranded DNA were evaluated. These results show that this region participates and allows for efficient recognition and excision of uracil within DNA. Notably, R86A AFUDG exhibited reduced activity for uracil removal only within double-stranded DNA, suggesting an importance in duplex disruption and extrusion of the base as part of the excision process. In addition, mutation of the [4Fe-4S](2+) cluster cysteine ligands at the ends of the pseudo-FCL to alanine reduced the uracil excision efficiency, suggesting the importance of anchoring the loop via coordination to the cluster. In contrast, K100A AFUDG exhibited enhanced uracil excision activity, providing evidence for the importance of the loop conformation and flexibility. Taken together, the results herein provide evidence that the pseudo-FCL motif is involved in DNA binding and catalysis, particularly in duplex DNA contexts. This work underscores the requirement of an ensemble of interactions, both distant and in proximity to the damaged site, for accurate and efficient uracil excision.

Dataset Information

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

Publications

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

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