Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.
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PMID:The genome of Melanoplus sanguinipes entomopoxvirus. 984 59

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.
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PMID:DNA repair enzyme uracil DNA glycosylase is specifically incorporated into human immunodeficiency virus type 1 viral particles through a Vpr-independent mechanism. 988 80

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM). Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the >10(6)-fold catalytic specificity of UDG. Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism. The first step (E + S left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM). The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 1200 s-1). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.
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PMID:Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase. 989 91

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.
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PMID:Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited. 1009 Feb 82

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.
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PMID:DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway. 1065 42

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.
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PMID:Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase. 1102 38

Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG). The binding of undamaged DNA to UDG results in decreased intensity of the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2'-beta-fluorodeoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-)(1) downshifts in the carbonyl normal modes. Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouridine analogue such that a change in hybridization at C1 occurs [Parikh, S. S., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueous solution, contributing significantly to catalysis. These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.
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PMID:Raman spectroscopy of uracil DNA glycosylase-DNA complexes: insights into DNA damage recognition and catalysis. 1105 77

The DNA repair enzyme uracil DNA glycosylase catalyzes the first step in the uracil base excision repair pathway, the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA. Here we report kinetic isotope effect (KIE) measurements that have allowed the determination of the transition-state structure for this important reaction. The small primary (13)C KIE (=1.010 +/- 0.009) and the large secondary alpha-deuterium KIE (=1.201 +/- 0.021) indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) there is significant sp(2) character at the anomeric carbon. Large secondary beta-deuterium KIEs were observed when [2'R-(2)H] = 1.102 +/- 0.011 and [2'S-(2)H] = 1.106 +/- 0.010. The nearly equal and large magnitudes of the two stereospecific beta-deuterium KIEs indicate strong hyperconjugation between the elongated glycosidic bond and both of the C2'-H2' bonds. Geometric interpretation of these beta-deuterium KIEs indicates that the furanose ring adopts a mild 3'-exo sugar pucker in the transition state, as would be expected for maximal stabilization of an oxocarbenium ion. Taken together, these results strongly indicate that the reaction proceeds through a dissociative transition state, with complete dissociation of the uracil anion followed by addition of water. To our knowledge, this is the first transition-state structure determined for enzymatic cleavage of the glycosidic linkage in a pyrimidine deoxyribonucleotide.
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PMID:Kinetic isotope effect studies of the reaction catalyzed by uracil DNA glycosylase: evidence for an oxocarbenium ion-uracil anion intermediate. 1108 52

The DNA repair enzyme uracil DNA glycosylase (UDG) is a powerful N-glycohydrolase that cleaves the glycosidic bond of deoxyuridine in DNA. We have investigated the role of substrate binding energy in catalysis by systematically dismantling the optimal substrate Ap(+1)UpA(-1)pA(-2) by replacing the nucleotides at the +1, -1, or -2 position with a tetrahydrofuran abasic site nucleotide (D), a 3-hydroxypropyl phosphodiester spacer (S), a phosphate monoester (p), or a hydroxyl group (h). Contrary to previous reports, the minimal substrate for UDG is 2'-deoxyuridine (hUh). UDG has a significant catalytic efficiency (CE) for hUh of 4 x 10(7) M(-1) [CE = (k(cat)/K(m))(1/k(non)), where k(non) is the rate of the spontaneous hydrolysis reaction of hUh at 25 degrees C]. Addition of +1 and -1 phosphate monoanions to form pUp increases k(cat)/K(m) by 45-fold compared to that of hUh. The k(cat)/K(m) for pUp, but not pU or Up, is found to decrease by 20-fold over the pH range of 6-9 with a pK(a) of 7.1, which is identical to the pK(a) values for deprotonation of the +1 and -1 phosphate groups determined by the pH dependence of the (31)P NMR chemical shifts. This pH dependence indicates that binding of the pUp tetraanion is disfavored, possibly due to unfavorable desolvation or electrostatic properties of the highly charged +1 and -1 phosphate groups. Addition of flexible hydroxypropyl groups to the +1 and -1 positions to make SpUpS increases k(cat)/K(m) by more than 10(5)-fold compared to that of hUh, which is a 20-fold greater effect than observed with rigid D substituents in these positions (i.e., DpUpD). The -2 phosphoester or nucleotide is found to increase the reactivity of trimer substrates with rigid furanose rings or nucleotides in the +1 and -1 positions by 1300-270000-fold (i.e., DpUpD --> DpUpDpA or ApUpA --> ApUpApA). In contrast, the -2 nucleotide provides only an 8-fold rate enhancement when appended to the substrate containing the more flexible +1 and -1 S substituents (SpUpS --> SpUpSpA). These context-dependent effects of a -2 nucleotide are interpreted in terms of a mechanism in which the binding energy of this "handle" is used drive the rigid +1 and -1 A or D substituents into their binding pockets, resulting in a net catalytic benefit of -4.3 to -7.5 kcal/mol. Taken together, these results systematically track how UDG uses distant site binding interactions to produce an overall four billion-fold increase in CE compared to that of the minimal substrate hUh.
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PMID:Reconstructing the substrate for uracil DNA glycosylase: tracking the transmission of binding energy in catalysis. 1141 25

Ion-pair reverse-phase high-performance liquid chromatography is presented as a versatile platform for the rapid analysis of nucleic acid modification reactions in a high-throughput manner. This system allows both sensitive and nonradioactive assays to be developed for a variety of nucleic acid modification reactions. Examples presented here include assays for telomerase, uracil DNA glycosylase, polynucleotide kinase, T4 DNA ligase, C5-DNA methyltransferases, and the mismatch endonuclease CEL I. However, this approach is not confined to these reactions. Indeed the ability to perform a variety of nonradioactive assays with throughput times of 10 min per sample in conjunction with automated data analysis software represents a significant improvement in analytical and preparative nucleic acid enzymology.
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PMID:High-throughput analysis of nucleic acid modification reactions using ion-pair reverse-phase high-performance liquid chromatography. 1181 99


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