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)

Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.
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PMID:Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding. 200 4

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.
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PMID:Biosynthesis of mammalian DNA ligase. 397 11

A composite plasmid pLS253 was constructed from pLS103 [carrying the Bacillus subtilis leucine genes on B. subtilis (natto) plasmid pLS28] and pHV14 [a recombinant plasmid composed of pBR322 and the staphylococcal R-plasmid pC194] employing BamHI endonuclease, T4 DNA ligase, and B. subtilis transformation. All the Leu+ Cmr transformants tested harbored not only pLS253 but also two smaller plasmids designated as pLS251 and pLS252. pLS253 DNA, when purified on an agarose gel, retained both Leu+ and Cmr transforming activities; however, in all the Leu+ Cmr transformants, the two smaller plasmids reappeared. pLS251 and pLS252 exhibited Leu+- or Cm4-transforming activity, respectively, and must have been derived from the pLS253 parent by an intramolecular recombination event, since the sum of the pLS251 and pLS252 DNAs represent the entire pLS253 genome. The recombination occurred between specific sites on the B. subtilis (natto) and Staphylococcus aureus plasmids. When the composite plasmid, pLS254, was constructed by BamHI cleavage of pLS251 and pLS252 followed by ligation, Leu+ Cmr transformants segregated two smaller plasmids which were indistinguishable from the original plasmids pLS103 and pHV14, respectively. They must have been derived from pLS254 through a reversal of the original recombination event. No intermolecular recombination between pLS251 and pLS252 DNA was detected. The recombination process was independent of recE function of the host cells, and its mechanism is discussed.
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PMID:A site-specific recE4-independent intramolecular recombination between Bacillus subtilis and Staphylococcus aureus DNAs in hybrid plasmids. 678 75

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

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic. Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi. In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.
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PMID:The role of leucine 191 of Escherichia coli uracil DNA glycosylase in the formation of a highly stable complex with the substrate mimic, ugi, and in uracil excision from the synthetic substrates. 1127 52

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.
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PMID:Mutational analysis of the base-flipping mechanism of uracil DNA glycosylase. 1222 Jan 89

NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI ((43)LXDG(46)). Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities. Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine. Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level. On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity. The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar. The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions. The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI. The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.
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PMID:Amino acid substitutions at position 43 of NaeI endonuclease. Evidence for changes in NaeI structure. 1251 52

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.
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PMID:Characterization of a temperature-sensitive DNA ligase from Escherichia coli. 1558 69

Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 degrees C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu(272) loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu(272) loop. The flexibility in this loop correlates well with the experimental k(cat)/K(m) values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments.
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PMID:Increased flexibility as a strategy for cold adaptation: a comparative molecular dynamics study of cold- and warm-active uracil DNA glycosylase. 1574 96

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.
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PMID:Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. 1659 52


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