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 cell cycle-dependent transcription factor, E2F-1, regulates the cyclin-like species of the DNA repair enzyme uracil-DNA glycosylase (UDG) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human UDG promoter sequences, that expression of E2F-1 activates the UDG promoter through several E2F sites. The major putative downstream site for E2F, located in the first exon, serves as a target for E2F-1/DP1 complex binding in vitro. We also provide evidence for the functional relationship between the cyclin-like UDG gene product and E2F. High levels of UDG expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of UDG in Saos 2 cells was observed to delay growth late in G1 phase and transiently arrest these cells from progressing into the S phase. This hypothetical model integrates one mechanism of DNA repair with the cell cycle control of gene transcription, likely through E2F. This implicates E2F as a multifunctional target for proteins and enzymes, possibly, responsive to DNA damage through the negative effect of UDG on E2F-mediated transcriptional activity.
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PMID:E2F-1 and a cyclin-like DNA repair enzyme, uracil-DNA glycosylase, provide evidence for an autoregulatory mechanism for transcription. 753 93

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.
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PMID:Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. 767

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.
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PMID:Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. 769 13

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

Uracil-DNA glycosylase encoded in many species functions as a DNA repair enzyme that removes uracil residues from DNA. To investigate the potential function of uracil-DNA glycosylase encoded by human herpes-virus 6 (HHV-6), we sequenced a DNA clone (pSTY09), identified an open reading frame of 765 bp and compared the putative amino acid sequence with other uracil-DNA glycosylases, by computer analysis. The amino acid sequence of HHV-6 had similarities to other uracil-DNA glycosylases, with the highest degree of similarity to those of human cytomegalovirus and Epstein-Barr virus. Two strongly conserved regions in uracil-DNA glycosylase of other species also existed in HHV-6. The gene product which was expressed in Escherichia coli demonstrated uracil-DNA glycosylase activity. This is the first report to identify and characterize the uracil-DNA glycosylase gene in HHV-6.
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PMID:Identification of human herpesvirus 6 uracil-DNA glycosylase gene. 807 33

Deinococcus radiodurans is the most radioresistant bacterium discovered to date. Recently it has been demonstrated that this organism contains the DNA repair enzyme uracil-DNA glycosylase and an apurinic/apyrimidinic (AP) endonuclease that may function as part of a DNA base excision repair pathway. We demonstrate here that a DNA deoxyribophosphodiesterase activity that acts on incised AP sites in DNA to remove deoxyribose-phosphate groups is found in lysates prepared from D. radiodurans cells. The partially purified activity was found to be smaller in size than the E. coli dRpase activity, with an estimated molecular weight of 25-30 kDa. In addition, an activity that recognizes and cleaves DNA containing thymine glycols was also detected, with a molecular weight of approximately 30 kDa. This enzyme may be analogous to the thymine glycol glycosylase/AP lyase endonuclease III of E. coli.
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PMID:DNA deoxyribophosphodiesterase and an activity that cleaves DNA containing thymine glycol adducts in Deinococcus radiodurans. 818 99

A 28.5 kDa catalytic fragment of the uracil-DNA glycosylase DNA repair enzyme from Herpes simplex virus type 1 (HSV-1) has been crystallized using protein from a highly expressing Escherichia coli clone of the Herpes simplex virus type 1 UL2 gene. The protein crystallizes at 12 mg/ml from 11% (w/v) polyethylene glycol 8000 at pH values in the range 6.8 to 7.0, in the presence of (NH4)2SO4. Long trigonal rods (0.08 mm x 0.08 mm x > 0.5 mm) diffract beyond 3.0 A using a laboratory source. The enzyme crystallizes in P3(1) (or P3(2)) a = 65.3 A, c = 49.0 A with a single molecule in the asymmetric unit and an estimated solvent content of 41% by volume.
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PMID:Crystallization and preliminary X-ray analysis of the uracil-DNA glycosylase DNA repair enzyme from herpes simplex virus type 1. 825 88

The predicted amino acid sequence of a human cDNA encoding uracil-DNA glycosylase activity shows striking similarity to the cyclin protein family. To characterize the expression of this DNA repair enzyme, we have isolated the corresponding genomic clone. This gene is contained within 4.2 kilobases and is composed of only two exons. Sequence analysis of the upstream region shows that it contains two cell cycle box (CCB) regulatory elements which are also found in yeast cyclin genes. Deletional analysis of the promoter reveals the presence of a repressor region located from position -812 to -603. An inverted CCB element (alpha-CCB) and an SP1-like binding site are contained within this region. When uracil-DNA glycosylase mRNA levels are examined in vivo, a 2-3-fold increase is associated with G1 phase in both HeLa S3 and WI38 cells. To examine the role of the 209-base pair repressor region in mediating cell cycle regulation, this fragment was used in gel shift assays with cellular extracts prepared from various stages of the cell cycle. Several specific complexes are formed during S and G2 phases which are not present during M and G1 phases. Two of the complexes are the result of alpha-CCB binding as they can be specifically disrupted by the addition of an oligonucleotide containing the alpha-CCB binding site. Immunoprecipitation studies reveal that uracil-DNA glycosylase protein levels are also elevated during G1 phase. Additionally, we show that the 36-kDa uracil-DNA glycosylase protein is turned over during the course of one cell cycle. These results demonstrate coordinate regulation of uracil-DNA glycosylase at both the transcriptional and the post-transcriptional levels as a function of the cell cycle.
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PMID:Cell cycle regulation of a human cyclin-like gene encoding uracil-DNA glycosylase. 841 33

Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes. Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V. Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.
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PMID:A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. 890 Feb 70

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.
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PMID:Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats. 939 4


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