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James T Stivers

Publications and source records attributed to James T Stivers.

At least 19 recordsLinked to original sources
PubMed2007

Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I.

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5C4+C3+T+2T+1p \N-1 using an active-site tyrosine nucleophile, thereby expelling a 5' hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3'-GGGAA-5' consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3' to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.

Base Sequence↗
PubMed2006

Mimicking damaged DNA with a small molecule inhibitor of human UNG2.

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

Binding Sites↗
PubMed2006

The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs.

Uracil DNA glycosylase (UNG) locates uracil and its structural congener thymine in the context of duplex DNA using a base flipping mechanism. NMR imino proton exchange measurements were performed on free and UNG-bound DNA duplexes in which a single thymine (T) was paired with a series of adenine analogues (X) capable of forming one, two, or three hydrogen bonds. The base pair opening equilibrium for the free DNA increased 55-fold as the number of hydrogen bonds decreased, but the opening rate constants were nearly the same in the absence and presence of UNG. In contrast, UNG was found to slow the base pair closing rate constants (kcl) compared to each free duplex by a factor of 3- to 23-fold. These findings indicate that regardless of the inherent thermodynamic stability of the TX pair, UNG does not alter the spontaneous opening rate. Instead, the enzyme holds the spontaneously expelled thymine (or uracil) in a transient extrahelical sieving site where it may partition forward into the enzyme active site (uracil) or back into the DNA base stack (thymine).

Base Pairing↗
PubMed2006

Major groove interactions of vaccinia Topo I provide specificity by optimally positioning the covalent phosphotyrosine linkage.

Vaccinia DNA topoisomerase (vTopo) is a prototypic eukaryotic type I topoisomerase that shows high specificity for nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5 C+4 C+3 T+2 T+1 p / N(-1). This reaction involves reversible transesterification where the active site tyrosine of the enzyme and a 5'-hydroxyl nucleophile of DNA compete for attack at the phosphoryl group. The finite lifetime of the covalent phosphotyrosine adduct allows the enzyme to relax multiple supercoils by rotation of the 5'-OH strand before the DNA backbone is religated. To dissect the nature of the unique sequence specificity, subtle modifications to the major groove of the GGGAA 5'-sequence of the nonscissile strand were introduced and their effects on each step of the catalytic cycle were measured. Although these modifications had no effect on noncovalent DNA binding (K(D)) or the rate of reversible DNA cleavage (k(cl)), significant decreases in the cleavage equilibrium (K(cl) = k(cl)/k(r)) arising from increased rates of 5'-hydroxyl attack (k(r)) at the phosphotyrosine linkage were observed. These data and other findings support a model in which major groove interactions are used to position the phosphotyrosine linkage relative to the mobile 5'-hydroxyl nucleophile. In the absence of native sequence interactions, the phosphotyrosine has a higher probability of encountering the 5'-hydroxyl nucleophile, leading to an enhanced rate of ligation and a diminished equilibrium constant for cleavage. By this unusual specificity mechanism, the enzyme prevents formation of stable covalent adducts at nonconsensus sites in genomic DNA.

Base Sequence↗
PubMed2006

Synthesis and high-throughput evaluation of triskelion uracil libraries for inhibition of human dUTPase and UNG2.

Human nuclear uracil DNA glycosylase (UNG2) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA. These enzymes are desirable targets for small molecule inhibitors given their roles in a wide range of biological processes ranging from chromosomal rearrangements that lead to cancer, viral DNA replication, and the formation of toxic DNA strand breaks during anticancer drug therapy. To accelerate the discovery of such inhibitors, we have developed a high-throughput approach for directed library synthesis and screening. In this efficient technology, a uracil-aldehyde ligand is covalently tethered to one position of a trivalent alkyloxyamine linker via an oxime linkage, and then the vacant linker positions are derivatized with a library of aldehydes. The resulting triskelion oximes were directly screened for inhibitory activity and the most potent of these showed micromolar binding affinities to UNG2 and dUTPase.

Aldehydes↗
PubMed2006

Linking uracil base excision repair and 5-fluorouracil toxicity in yeast.

5-fluorouracil (5-FU) is a widely used anticancer drug that disrupts pyrimidine nucleotide pool balances and leads to uracil incorporation in DNA, which is then recognized and removed by the uracil base excision repair (BER) pathway. Using complementary biochemical and genetic approaches we have examined the role of uracil BER in the cell killing mechanism of 5-FU. A yeast strain lacking the enzyme uracil DNA glycosylase (Ung1), which excises uracil from the DNA backbone leaving an abasic site, showed significant protection against the toxic effects of 5-FU, a G1/S cell cycle arrest phenotype, and accumulated massive amounts of U/A base pairs in its genome (approximately 4% of T/A pairs were now U/A). A strain lacking the major abasic site endonuclease of Saccharomyces cerevisiae (Apn1) showed significantly increased sensitivity to 5-FU with G2/M arrest. Thus, efficient processing of abasic sites by this enzyme is protective against the toxic effects of 5-FU. However, contrary to expectations, the Apn1 deficient strain did not accumulate intact abasic sites, indicating that another repair pathway attempts to process these sites in the absence Apn1, but that this process has catastrophic effects on genome integrity. These findings suggest that new strategies for chemical intervention targeting BER could enhance the effectiveness of this widely used anticancer drug.

Antimetabolites, Antineoplastic↗
PubMed2005

Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development.

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.

Aldehydes↗
PubMed2005

Novel and specific inhibitors of a poxvirus type I topoisomerase.

Vaccinia DNA topoisomerase (vTopo) is a prototypic pox virus family topoisomerase that shares extensive structural and mechanistic properties with the human type IB enzyme (hTopo) and is important for viral replication. Despite their far-reaching similarities, vTopo and hTopo have surprisingly distinct pharmacological properties. To further exploit these differences, we have developed recently the first high-throughput screen for vTopo, which has allowed rapid screening of a 1990-member small-molecule library for inhibitors. Using this approach, 21 compounds were identified with IC(90) values less than 10 muM, and 19 of these were also found to inhibit DNA supercoil relaxation by vTopo. Four of the most potent compounds were completely characterized and are structurally novel topo I inhibitors with efficacies at nanomolar concentrations. These inhibitors were highly specific for vTopo, showing no inhibition of the human enzyme even at 500- to 2000-fold greater concentrations. We describe a battery of efficient experiments to characterize the unique mechanisms of these vTopo inhibitors and discuss the surprising promiscuity of this enzyme to inhibition by structurally diverse small molecules.

Antiviral Agents↗
PubMed2005

Catalytic phosphoryl interactions of topoisomerase IB.

The reversible nucleophilic substitution reaction catalyzed by the vaccinia virus type IB topoisomerase has been investigated by measuring the equilibrium and rate effects of stereospecific sulfur substitution at the two nonbridging oxygen atoms of the attacked phosphodiester group. An energetic analysis of the combined effects of sulfur substitution and site-directed mutagenesis of active site residues of the enzyme has identified enzyme interactions with each oxygen in the ground state and transition state. We use these findings in combination with previous structural and 5'-bridging sulfur substitution results to deduce the web of enzymatic interactions with the nonbridging oxygens as well as the 5'-hydroxyl leaving group. A key finding is the central role of Arg130, which forms electrostatic interactions with both nonbridging oxygens and the 5'-leaving group.

Amino Acid Substitution↗
PubMed2005

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase.

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.

Animals↗
PubMed2005

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.

Base Pairing↗
PubMed2004

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.

Amino Acid Substitution↗
PubMed2004

Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.

Anisotropy↗
PubMed2004

Dynamic opening of DNA during the enzymatic search for a damaged base.

Uracil DNA glycosylase (UDG) removes uracil from U.A or U.G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.

Base Sequence↗
PubMed2004

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.

Base Pairing↗
PubMed2004

The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase.

The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA. We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached. We find that the bipartite mimics of TS2 are superior to the TS1 mimics. These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.

Amides↗