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An attempt is made to show that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway. Structural models of the metastable state that are consistent with the REDOR NMR measurements are suggested.

The effects of a C1'-oxidized abasic site incorporated at a defined site in single-stranded plasmid were examined in SOS polymerase-proficient and -deficient Escherichia coli. In the overall context of their effects on E.coli, it appears that abasic lesions are instructive.

The synthesis of the stable, water-soluble receptor contains a 2,6-bis(glycylamino)pyridine group as a structural mimic of the active site in uracil DNA glycosylase (UDG) understand the functional role of UDG which pulls unwanted uracil into it active site through hydrogen bonding and [pi]-[pi] stacking interactions. The obtained results provide insight into the binding mechanism of uracil and its derivatives in DNA, by DNA repair enzymes through hydrogen bonding and [pi]-[pi] stacking interactions.

The effect of Fapy.dG on replication in Escherichia coli is analyzed by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in local sequence contexts. The results have shown that a low dA misincorporation frequency opposite Fapy.dG and inefficient extension of a Fapy.dG:dA base pair have worked synergistically to minimize the levels of G -> T transversions.

The nucleoside triphosphates of N6-(2-deoxy-[alpha],[beta],D--erthro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTAP) and its C-nucleoside analogue ([beta]-C-Fapy.dGTP) are synthesized. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP.

A study measured deoxyribonucleic acid (DNA) base pair opening and closing rates in the absence and presence of uracil DNA glycolase (UNG) for a series of increasingly destabilized thymine base pairs (TX) that contain one, two, or three hydrogen bonds. Comparison of the measured opening rates ([k.sub.op]) for each [T.sup.6]X base pair in the free and UNG-bound DNA indicates that UNG does not actively accelerate base pair opening.

An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine or 5'-deoxycytidine are prepared and employed in oligonucleotide synthesis.

Substrate and product structures are compared with the previous structure of the intermediate determined by kinetic isotope effects. The study reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis.

Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by 1H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired. [ABSTRACT FROM AUTHOR]

Through middle-temperature solvothermal reactions of CoCl2·6H2O with the rigid-angled ligand 3-(2′-pyridyl)-5-(4″'-pyridyl)-1,2,4-triazole (Hdpt24), we obtained the three cobalt complexes {[Co(dpt24)2)]3·4DMF·1.5H2O}n (1), {[Co(dpt24)2)]2·H2O}n (2), and Co(dpt24)2(Hdpt24)·H2O] (4) at N,N-dimethylformamide (DMF)/H2O volume ratios of 9:1, 1:1, and 0:1, respectively. Interestingly, 1 underwent transformations into 2, {[Co(dpt24)2]·0.5DMF}n (3), and 4 when treated with DMF/H2O at volume ratios of 1:1, 1:9, and 0:1, respectively. Moreover, 3 and 4 converted back to 1 in 9:1 DMF/H2O and to 2 in 1:1 DMF/H2O; 3 transformed into 4 in H2O and vice versa in 1:9 DMF/H2O. Structurally, 1 is a three-dimensional (3D) 2-fold interpenetrating distorted NbO-type complex, 2 possesses a two-dimensional layer metal-organic framework, 3 is a 3D 2-fold interpenetrating typical NbO-type complex, and 4 is a wheel-shaped mononuclear neutral complex. This approach, using a mixed solvent's component ratio to direct the syntheses and conversions of four cobalt complexes, provides unprecedented control for crystal engineering. [ABSTRACT FROM AUTHOR]

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases. [ABSTRACT FROM AUTHOR]

Fapy dG (N6-(2-deoxy-α,β-D-erythropentofuranosyl)-2 ,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy·dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy·dG, which does not require reverse phosphoramidites, is described. Fapy·dG containing dinucleotide phosphoramidites containing 5′-thymidine (ha) or 5′-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS. [ABSTRACT FROM AUTHOR]

Discusses the mechanistic perspective on the chemistry of DNA repair glycosylases. Mechanisms for involvement of the furanose sugar in nonenzymatic glycosidic bond cleavage; Pathway for finding a damaged base in genomic DNA; Discussion of enzymatic base flipping.

The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxvribose (1-azadR[sup +]) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k[sub cat]/K[sub m] for the series of MeP-substituted substrates against log K[sub D] for binding of the 1-aza-dR[sup +] inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR[sup +], and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA. [ABSTRACT FROM AUTHOR]

Examines the base-flipping mutations of uracil DNA glycosylase using pyrene nucleotide wedge. Emphasis on the kinetic effects of mutation; Link between amino acid side chain and energetic consequence; Understanding the functional role of amino acid.

Describes the mutational analysis of the base-flipping mechanism of uracil DNA glycosylase. Promotion of uracil flipping; Decrease of the intensities of DNA base Raman bands; Removal of enzymatic side chains.

Investigates uracil DNA glycosylase (UDG) suppression by oxacarbenium ion mimic. Properties of UDG; Inhibition of alpha and beta-glycosidases by glycosyl cation; Mechanism of UDG inhibition.

Studies how uracil DNA glycosylase (UDG) uses distant site binding interactions to produce an overall increase in catalytic efficiency compared to that of the minimal substrate 2'-deoxyuridine. Nucleotide replacements; Rate of the spontaneous hydrolysis reaction; pH dependence; Addition of hydroxypropyl groups; Context-dependent effects of a -2 nucleotide.

Examines the role of serine pinching in damaged site recognition and catalysis by uracil DNA glycosylase. Effects of 5' pinching on glycosidic bond cleavage; Details of the damaging effects of S88A and S192G pinching mutation on kinetic parameters; Energy analysis of nonbridging phosphorothioate substitution with the mutation.