Repair Enzyme Uracil DNA Glycosylase Research Articles

Fluorometric determination of the activity of uracil-DNA glycosylase by using graphene oxide and exonuclease I assisted signal amplification.

The base-excision repair enzyme uracil-DNA glycosylase (UDG) plays a crucial role in the maintenance of genome integrity. The authors describe a fluorometric method for the detection of the activity of UDG. It is making use of (a) a 3'-FAM-labeled hairpin DNA probe with two uracil deoxyribonucleotides in the self-complementary duplex region of its hairpin structure, (b) exonuclease I (Exo I) that catalyzes the release of FAM from the UDG-induced stretched ssDNA probe, and (c) graphene oxide that quenches the greenFAM fluorescence of the intact hairpin DNA probe in the absence of UDG. If Exo I causes the release of FAM from the hairpin DNA probe, the fluorescence peakingat 517nm is turned off in the absence of UDG but turned on in its presence. The resulting assay has a wide linear range (0.008 to 1U·mL-1) and a detection limit as low as 0.005U·mL-1. It has good specificity for UDG over potentially interfering enzymes and gave satisfactory results when applied to biological samples. Conceivably, the method may be used in a wide range of applications such as in diagnosis, drug screening, and in studying the repair of DNA lesions. Graphical abstract Schematic presentation of a fluorometric strategy for detection of the activity of uracil-DNA glycosylase by using on graphene oxide and exonuclease I assisted signal amplification.

Backbone resonance assignment of the human uracil DNA glycosylase-2.

The HIV-1 viral protein R (Vpr) is incorporated into virus particle during budding suggesting that its presence in the mature virion is required in the early steps of the virus life cycle in newly infected cells. Vpr is released into the host cell cytoplasm to participate to the translocation of the preintegration complex (PIC) into the nucleus for integration of the viral DNA into the host genome. Actually, Vpr plays a key role in the activation of the transcription of the HIV-1 long terminal repeat (LTR), mediates cell cycle arrest in G2 to M transition, facilitates apoptosis and controls the fidelity of reverse transcription. Moreover, Vpr drives the repair enzyme uracil DNA glycosylase (UNG2) towards degradation. UNG2 has a major role in "Base excision repair" (BER) whose main function is to maintain genome integrity by controlling DNA uracilation. The interaction of Vpr with the cellular protein UNG2 is a key event in various stages of retroviral replication and its role remains to be defined. We have performed the structural study of UNG2 by NMR and we report its (1HN, 15N, 13Cα, 13Cβ and 13C') chemical shift backbone assignment and its secondary structure in solution as predicted by TALOS-N. We aim to determine with accuracy by NMR, the residues of UNG2 interacting with Vpr, characterize their interaction and use the local structure of UNG2 and its interface with Vpr to propose potential ligands disturbing this interaction.

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.

Multiple microRNAs may regulate the DNA repair enzyme uracil-DNA glycosylase

Human nuclear uracil-DNA glycosylase UNG2 is essential for post-replicative repair of uracil in DNA, and UNG2 protein and mRNA levels rapidly decline in G2/M phase. Previous work has demonstrated regulation of UNG2 at the transcriptional level, as well as by protein phosphorylation and ubiquitylation. UNG2 mRNA, encoded by the UNG gene, contains a long 3′untranslated region (3′UTR) of previously unknown function. Here, we demonstrate that several conserved regions in the 3′UTR are potential seed sites for microRNAs (miRNAs), such as miR-16, miR-34c, and miR-199a. Our results show that these miRNAs down-regulate UNG activity, UNG mRNA, and UNG protein levels. Down-regulation was dependent on the 3′UTR, indicating that the miRNAs directly target the conserved seed sites in the 3′UTR. These results add miRNAs as a new modality to UNG's increasing list of complex regulatory mechanisms.

Abstract 4696: Expression of the DNA repair protein UNG predicts lung cancer sensitivity to pemetrexed

Abstract Uracil misincorporation is a direct consequence of TS inhibition by the multi-target antifolate, pemetrexed. The DNA base excision repair (BER) enzyme Uracil DNA glycosylase (UNG) is the major glycosylase responsible for the removal of misincorporated genomic uracil. We have previously illustrated that human colon cancer cells (DLD1) with engineered stable somatic knockout of UNG expression are hypersensitive to pemetrexed owing to the cytotoxicity of genomic uracil accumulation. Here, we examined the relationship between the expression of UNG and the pemetrexed sensitivity in a small panel of lung cancer cell lines (n∼10). The levels of UNG were measured by western blot (for protein) and RT-PCR (for mRNA). Results revealed that UNG mRNA expression is well correlated with protein expression in all cell lines. Moreover, there was a wide range of distribution of UNG levels in the lung cancer cell lines evaluated. UNG cutting assay and AP site measurements confirm that these differences in UNG expression result in varied potential for BER activation and subsequent AP site formation. To determine the role of this variation in UNG expression in pemetrexed cytotoxicity, colony survival studies were performed. In general, cell lines with higher UNG expression were more resistant to pemetrexed. H460 cells having 7-fold lower expression of UNG mRNA than H1975 cells are >5 fold more sensitive to pemetrexed than H1975 cells (H460 IC50 = 200nM; H1975 IC50 = >1000nM). Analysis of tissue microarray data in Oncomine confirms variations in UNG expression in primary human lung cancer samples. Moreover, small cell lung cancer and squamous cell lung cancer, both notoriously resistant to pemetrexed, have significantly increased median UNG expression compared to all other lung cancer subtypes (1.5-fold greater UNG expression p<0.005). These data motivate future evaluation of UNG as a clinical predictor of pemetrexed response using lung cancer biopsy specimens. Together, our results clearly indicate that the BER protein, UNG is differentially expressed in lung cancer cells. The inherent variation in UNG expression may have particular impact on our ability to predict the therapeutic efficacy of pemetrexed in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4696. doi:1538-7445.AM2012-4696

Cosolute Paramagnetic Relaxation Enhancements Detect Transient Conformations of Human Uracil DNA Glycosylase (hUNG)

The human DNA repair enzyme uracil DNA glycosylase (hUNG) locates and excises rare uracil bases that arise in DNA from cytosine deamination or through dUTP incorporation by DNA polymerases. Previous NMR studies of hUNG have revealed millisecond time scale dynamic transitions in the enzyme-nonspecific DNA complex, but not the free enzyme, that were ascribed to a reversible clamping motion of the enzyme as it scans along short regions of duplex DNA in its search for uracil. Here we further probe the properties of the nonspecific DNA binding surface of {(2)H(12)C}{(15)N}-labeled hUNG using a neutral chelate of a paramagnetic Gd(3+) cosolute (Gd(HP-DO3A)). Overall, the measured paramagnetic relaxation enhancements (PREs) on R(2) of the backbone amide protons for free hUNG and its DNA complex were in good agreement with those calculated based on their relative exposure observed in the crystal structures of both enzyme forms. However, the calculated PREs systematically underestimated the experimental PREs by large amounts in discrete regions implicated in DNA recognition and catalysis: active site loops involved in DNA recognition (268-274, 246-250), the uracil binding pocket (143-148, 169-170), a transient extrahelical base binding site (214-216), and a remote hinge region (129-132) implicated in dynamic clamping. These reactive hot spots were not correlated with structural, hydrophobic, or solvent exchange properties that might be common to these regions, leaving the possibility that the effects arise from dynamic sampling of exposed conformations that are distinct from the static structures. Consistent with this suggestion, the above regions have been previously shown to be flexible based on relaxation dispersion measurements and course-grained normal-mode analysis. A model is suggested where the intrinsic dynamic properties of these regions allows sampling of transient conformations where the backbone amide groups have greater average exposure to the cosolute as compared to the static structures. We conclude that PREs derived from the paramagnetic cosolute reveal dynamic hot spots in hUNG and that these regions are highly correlated with substrate binding and recognition.

Implications of Alternative Substrate Binding Modes for Catalysis by Uracil-DNA Glycosylase: An Apparent Discrepancy Resolved

A theoretical study showed that the base excision repair enzyme uracil-DNA glycosylase (UDG) exploits electrostatic interactions with backbone phosphate groups in the substrate for catalysis. Although experiments performed to test the calculated results confirmed the predicted importance of the -2, -1, and +1 phosphate groups, there was an apparent disagreement with regard to the +2 phosphate group. The calculations indicated that it made an important contribution, while experimentally, the effect of its deletion or neutralization was small. The +2 phosphate group interacts directly with an active site histidine (H148 in humans) in the crystal structure of UDG in complex with double-stranded (ds) DNA. We previously calculated that H148 has a strong anticatalytic effect due to its protonation, and here we use alchemical free energy simulations to estimate its site-specific pKa. We find that it is positively charged over the entire experimental pH range (4-10), so its deprotonation cannot compensate for deletion or neutralization of the +2 phosphate group. The free energy simulations are facilitated by an efficient charge-scaling procedure that allows quantitative correction for the implicit treatment of solvent far from the active site; improvements are made to that method to account carefully for differences in the truncation of electrostatic interactions in the contributing molecular-mechanical and continuum-electrostatic approaches. Additional simulations are used to demonstrate that the +2 phosphate group is fully solvent exposed in complexes with single-stranded DNA substrates like those used in the experiments. In contrast, it is well-structured and buried in the dsDNA complex used in the original simulations. Differences in solvent shielding thus account for the apparent lack of an effect observed experimentally upon neutralization or deletion of this group.

E2F1 Modulates p38 MAPK Phosphorylation via Transcriptional Regulation of ASK1 and Wip1

E2F1 Modulates p38 MAPK Phosphorylation via Transcriptional Regulation of ASK1 and Wip1

Fluorodeoxyuridine Modulates Cellular Expression of the DNA Base Excision Repair Enzyme Uracil-DNA Glycosylase

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G(1) and is degraded during the S to G(2) phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd. Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy.

The Origins of High-Affinity Enzyme Binding to an Extrahelical DNA Base

Base flipping is a highly conserved strategy used by enzymes to gain catalytic access to DNA bases that would otherwise be sequestered in the duplex structure. A classic example is the DNA repair enzyme uracil DNA glycosylase (UDG) which recognizes and excises unwanted uracil bases from DNA using a flipping mechanism. Previous work has suggested that enzymatic base flipping begins with dynamic breathing motions of the enzyme-bound DNA substrate, and then, only very late during the reaction trajectory do strong specific interactions with the extrahelical uracil occur. Here we report that UDG kinetically and thermodynamically prefers substrate sites where the uracil is paired with an unnatural adenine analogue that lacks any Watson-Crick hydrogen-bonding groups. The magnitude of the preference is a striking 43000-fold as compared to an adenine analogue that forms three H-bonds. Transient kinetic and fluorescence measurements suggest that preferential recognition of uracil in the context of a series of incrementally destabilized base pairs arises from two distinct effects: weak or absent hydrogen bonding, which thermodynamically assists extrusion, and, most importantly, increased flexibility of the site which facilitates DNA bending during base flipping. A coupled, stepwise reaction coordinate is implicated in which DNA bending precedes base pair rupture and flipping.

Recognition of an Unnatural Difluorophenyl Nucleotide by Uracil DNA Glycosylase

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.

Detection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides.

A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme-dependent label release (in case of glycosylase and endonuclease), or non-release (in case of methyltransferase) into solution from end-labeled DNA immobilized on solid support (CPG or Tenta Gel S-NH2). The assay has been validated for monitoring activity of repair enzyme uracil-DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine-5)-DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase-immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary.

Abasic site stabilization by aromatic DNA base surrogates: High-affinity binding to a base-flipping DNA-methyltransferase

Abstract DNA-methyltransferases catalyze the sequence-specific transfer of the methyl group of S-adenosylmethionine to target bases in genomic DNA. For gaining access to their target embedded within a double-helical structure, DNA-methyltransferases (DNA-MTases) rotate the target base out of the DNA helix. This base-flipping leads to the formation of an apparent abasic site. MTases such as cytosine-specific MHhaI and MHaeIII and also the repair enzyme uracil DNA glycosylase (UDG) insert amino acid side chains into the opened space and/or rearrange base-pairing. The adenine-specific DNA MTase MTaqI binds without amino acid insertion. This binding mode allows for a substitution of the orphaned thymine with larger DNA base surrogates without steric interference by inserted amino acid side chains. DNA containing pyrenyl, naphthyl, acenaphthyl, and biphenyl residues was tested in MTaqI binding studies. The synthesis of DNA building blocks required the formation of a C-glycosidic bond, which was established by using protected 1-chloro-2-deoxy- ribose as glycosyl donor and organocuprates as glycosyl acceptors. It is shown that all of the base surrogates enhanced the binding affinity to MTaqI. Incorporation of pyrene increased the binding affinity by a factor of 400. Interestingly, there is a correlation between the observed order of dissociation constants and the ability of a base surrogate to stabilize abasic sites in model duplexes.

Site-Specific DNA Damage Recognition by Enzyme-Induced Base Flipping

One of the most interesting enigmas in enzymatic DNA recognition is the mechanism by which damaged bases buried in the DNA base stack are detected and subsequently excised. The essential enzyme combatants in this never-ending battle against genomic instability, the DNA repair glycosylases (Fig. 1A), must act specifically against a variety of base lesions that arise from oxidative reactions, deamination events, and alkylating agents, without also mistakenly removing normal nucleobases (1). This is a high stakes game: the cost of being slow is irreversible damage to the coding content of the genome, and the cost of being sloppy is the indiscriminate introduction of toxic abasic sites in DNA (2–4). Because many damaged bases are nonperturbing to the duplex structure and may differ from normal bases by only a single atom change, an extraordinary and highly conserved recognition mechanism has evolved to accomplish this task. This mechanism has been termed “base flipping,” and involves the dramatic rotation of an entire damaged base and sugar from the DNA duplex using binding forces imposed by the enzyme (Fig. 1B) (5–7). The problem facing these enzymes is both enormous and unique. Consider that a damaged base may compose only one out of 107 base pairs in the human genome (8, 9). Thus, a crude estimate of the specificity for excision of a specific damaged base over a normal base of (kcat⧸Km)specific⧸(kcat⧸Km)nonspecific ≥ 107 may be assumed. Although such issues as increased accessibility of damaged vs normal bases in the densely packed chromatin may account for some of this apparently large specificity, it is difficult to dismiss the magnitude of the discrimination achieved by the recognition mechanisms of these enzymes. Equally remarkable is that specificity is achieved without forming sequence specific contacts with the DNA substrate. Indeed, any specificity mechanism that arose during natural selection involving sequence specific interactions would have been selected against, because this mechanism compromises the ability of the organism to execute unbiased repair of DNA lesions regardless of the sequence context. Accordingly, DNA glycosylases present a unique evolutionary solution to site recognition that is fundamentally different from that of restriction enzymes, repressor proteins, or transcription factors. Structural and other biophysical studies over the last four years have begun to reveal the basis for DNA glycosylase specificity and the critical role of DNA base flipping (5, 10–16). The system that has advanced most rapidly in this time span is that of the DNA repair enzyme uracil DNA glycosylase. This enzyme removes unwanted uracil residues in DNA that have arisen by spontaneous deamination of cytosine, or by misincorporation of dUTP during DNA replication (17). UDG has provided an experimentally tractable system to investigate the multistep process of base flipping, using a wide range of experimental approaches. The findings in this system should have widespread applicability given the highly conserved structural features of enzymes that flip bases (Fig. 1B). The general questions that have driven our research in base flipping over the last few years also direct the course of this chapter. To begin, we ask why has base flipping evolved as the sole mechanism for recognition and catalysis by DNA glycosylases? Second, what are the natures of the energetic barriers that an enzyme must overcome to extricate a base from its position in the DNA duplex? And finally, what temporal events occur during enzymatic base flipping, and how do the various steps contribute to catalytic specificity? We are continuing to develop new experimental tools to investigate base flipping, and we present these new approaches in the hope that they become generally useful in the investigation of other enzymes that flip bases.

Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.

Rational Engineering of a DNA Glycosylase Specific for an Unnatural Cytosine:Pyrene Base Pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a “preflipped” cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.

A charge-scaling method to treat solvent in QM/MM simulations

We present a method to treat the solvent efficiently in hybrid quantum mechanical/molecular mechanical simulations of chemical reactions in enzymes. The method is an adaptation of an approach developed for molecular-mechanical free-energy simulations. The charges of each of the exposed ionizable groups are scaled, and the system is simulated in the presence of a limited number of explicit solvent molecules to obtain a reasonable set of structures. Continuum electrostatics methods are then used to correct the energies. Variations in the procedure are discussed with an emphasis on modifications from the original protocol. We illustrate the method by applying it to the study of a hydrolysis reaction in a highly charged system comprising a complex between the base excision repair enzyme uracil-DNA glycosylase and double-stranded DNA. The resulting adiabatic reaction profile is in good agreement with experiment, in contrast to that obtained without scaling the charges.

Powering DNA repair through substrate electrostatic interactions.

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-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), 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.

Roles of uracil-DNA glycosylase and dUTPase in virus replication.

Herpesviruses and poxviruses are known to encode the DNA repair enzyme uracil-DNA glycosylase (UNG), an enzyme involved in the base excision repair pathway that specifically removes the RNA base uracil from DNA, while at least one retrovirus (human immunodeficiency virus type 1) packages cellular UNG into virus particles. In these instances, UNG is implicated as being important in virus replication. However, a clear understanding of the role(s) of UNG in virus replication remains elusive. Herpesviruses, poxviruses and some retroviruses encode dUTPase, an enzyme that can minimize the misincorporation of uracil into DNA. The encoding of dUTPase by these viruses also implies their importance in virus replication. An understanding at the molecular level of how these viruses replicate in non-dividing cells should provide clues to the biological relevance of UNG and dUTPase function in virus replication.

Mutational analysis of the base-flipping mechanism of uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952-963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.