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)

Mex+ human lymphoma cell lines contain O6-methylguanine-DNA methyltransferase, a DNA repair enzyme that undergoes suicide inactivation on interaction with its substrate. The cells are therefore competent to remove the alkylation lesion O6-methylguanine from their DNA. However, several repair-deficient lymphoma cell lines (Mex-) are also known. It is shown here that Mex+ cells can be converted temporarily to a Mex- phenotype by growth in nontoxic concentrations of free O6-methylguanine. The depletion of methyltransferase activity is not a result of O6-methylguanine incorporation into DNA and subsequent demethylation by the enzyme. It is proposed that O6-methylguanine is mistakenly incorporated into tRNA molecules by means of a post-transcriptional ribosyl transfer reaction. The demethylation of such bases in tRNA has been demonstrated by using bacterial and human DNA repair enzymes. The existence of such a subversive repair of a methylated base in tRNA raises the possibility of competition between DNA and RNA for cellular DNA repair enzymes. Furthermore, it is proposed that the known aberrant methylation of tRNA in certain transformed cells, together with subversive tRNA repair, could account for the Mex- phenotype.
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PMID:Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair. 386 Aug 61

The total synthesis of the suppressor tRNA gene (ssu1) containing a 51-base pair long "natural" promoter has previously been reported. In this paper, we describe the synthesis and characterization of two genes (ssu3 and ssu4) in which the promoter sequence present in the parent ssu1 gene has been altered. The modifications, introduced as a part of the study of structure-function relationships, were placed in the two known prominent regions of homology. The change introduced in ssu3 was a G:C to A:T transition at -10 nucleotide, while the two changes incorporated in ssu4 involved a G:C to A:T transition at -36 nucleotide and a G:C to T:A transversion at -38 nucleotide. The promoters containing the modifications were constructed by T4-polynucleotide ligase joining of the appropriate chemically synthesized oligonucleotides. These promoters then replaced the promoter region of the cloned and previously synthesized ssu2 gene by utilization of the HindIII site present at the junction of the promoter and the structural gene. The genes containing the modified promoters have been cloned into the plasmid vector pBR322 by insertion into the unique Eco RI site. The DNA sequencing of the cloned genes now described as well as that of the previously cloned and chemically synthesized genes (ssu1 and ssu2) provide direct confirmation of the accuracy of the synthetic work (chemical and enzymatic) at all stages.
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PMID:The synthesis and cloning of two tyrosine suppressor tRNA genes with altered promoter sequences. 626 4

To elucidate the molecular mechanism by which poly(ADP-ribose) participates in DNA excision repair, we examined the effect of poly(ADP-ribose) on DNA ligase activity in DNA/histone and reconstituted chromatin systems. The ligase activity was markedly inhibited by histones; the inhibition varied depending on histone subfraction and DNA/histone ratio. Poly(ADP-ribose), either exogenous or synthesized in situ by poly(ADP-ribose) synthetase, reversed this inhibition by histone almost completely. This effect was specific for poly(ADP-ribose); polyanions such as mRNA, rRNAs, tRNA, and synthetic poly(A) were less effective or ineffective. The ligase activity with reconstituted chromatin as the substrate was about half of that with free DNA whereas the activities with these two substrates were almost the same in the presence of poly(ADP-ribose) synthesized in situ. The polymers synthesized under these conditions were exclusively bound to the synthetase. Together with our previous finding that the enzyme is the main acceptor of the polymer in DNA-damaged cells, these results suggest that poly(ADP-ribose) in the synthetase-bound form counteracts inhibition by histones and activates DNA ligase to rejoin DNA strands in polynucleosomal structures.
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PMID:Activation of DNA ligase by poly(ADP-ribose) in chromatin. 640 17

The molecular mechanism by which poly(ADP-ribose) participates in DNA repair was investigated using purified DNA ligase in DNA-histone systems. The ligase activity was markedly inhibited by histones; the inhibition was greater than 80% with histone H1 at concentrations equal to DNA. This inhibition was reversed efficiently by poly(ADP-ribose), either added exogenously or synthesized in situ with poly(ADP-ribose) synthetase. The reversal effect was specific for poly(ADP-ribose); other polyanions such as mRNA, rRNA's, tRNA, and synthetic poly(A) were less effective or totally ineffective. The poly(ADP-ribose) effect appeared to be caused by binding to histones and decreasing DNA-histone interactions. Poly(ADP-ribose) also had high affinity for DNA ligase. These observations, together with the findings of absolute dependence of poly(ADP-ribose) synthetase activity on DNA strand ends and extensive automodification of the synthetase in DNA-damaged cells, suggested a possible mechanism of poly(ADP-ribose) action in DNA repair, in which auto-modified poly(ADP-ribose) synthetase serves as a link between DNA damage and activation of DNA ligase.
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PMID:Inhibition of DNA ligase activity by histones and its reversal by poly(ADP-ribose). 665 29

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.
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PMID:Recombinant photoreactive tRNA molecules as probes for cross-linking studies. 753 27

tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.
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PMID:Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme. 766 16

Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression.
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PMID:The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA. 793 71

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.
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PMID:Efficient gene tagging in Arabidopsis thaliana using a gene trap approach. 935 17

Homologs of the eukaryotic DNA-end-binding protein Ku were identified in several bacterial and one archeal genome using iterative database searches with sequence profiles. Identification of prokaryotic Ku homologs allowed the dissection of the Ku protein sequences into three distinct domains, the Ku core that is conserved in eukaryotes and prokaryotes, a derived von Willebrand A domain that is fused to the amino terminus of the core in eukaryotic Ku proteins, and the newly recognized helix-extension-helix (HEH) domain that is fused to the carboxyl terminus of the core in eukaryotes and in one of the Ku homologs from the Actinomycete Streptomyces coelicolor. The version of the HEH domain present in eukaryotic Ku proteins represents the previously described DNA-binding domain called SAP. The Ku homolog from S. coelicolor contains a distinct version of the HEH domain that belongs to a previously unnoticed family of nucleic-acid-binding domains, which also includes HEH domains from the bacterial transcription termination factor Rho, bacterial and eukaryotic lysyl-tRNA synthetases, bacteriophage T4 endonuclease VII, and several uncharacterized proteins. The distribution of the Ku homologs in bacteria coincides with that of the archeal-eukaryotic-type DNA primase and genes for prokaryotic Ku homologs form predicted operons with genes coding for an ATP-dependent DNA ligase and/or archeal-eukaryotic-type DNA primase. Some of these operons additionally encode an uncharacterized protein that may function as nuclease or an Slx1p-like predicted nuclease containing a URI domain. A hypothesis is proposed that the Ku homolog, together with the associated gene products, comprise a previously unrecognized prokaryotic system for repair of double-strand breaks in DNA.
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PMID:Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. 1148 77

Alpha (alpha) proteobacteria comprise a large and metabolically diverse group. No biochemical or molecular feature is presently known that can distinguish these bacteria from other groups. The evolutionary relationships among this group, which includes numerous pathogens and agriculturally important microbes, are also not understood. Shared conserved inserts and deletions (i.e., indels or signatures) in molecular sequences provide a powerful means for identification of different groups in clear terms, and for evolutionary studies (see www.bacterialphylogeny.com). This review describes, for the first time, a large number of conserved indels in broadly distributed proteins that are distinctive and unifying characteristics of either all alpha-proteobacteria, or many of its constituent subgroups (i.e., orders, families, etc.). These signatures were identified by systematic analyses of proteins found in the Rickettsia prowazekii (RP) genome. Conserved indels that are unique to alpha-proteobacteria are present in the following proteins: Cytochrome c oxidase assembly protein Ctag, PurC, DnaB, ATP synthase alpha-subunit, exonuclease VII, prolipoprotein phosphatidylglycerol transferase, RP-400, FtsK, puruvate phosphate dikinase, cytochrome b, MutY, and homoserine dehydrogenase. The signatures in succinyl-CoA synthetase, cytochrome oxidase I, alanyl-tRNA synthetase, and MutS proteins are found in all alpha-proteobacteria, except the Rickettsiales, indicating that this group has diverged prior to the introduction of these signatures. A number of proteins contain conserved indels that are specific for Rickettsiales (XerD integrase and leucine aminopeptidase), Rickettsiaceae (Mfd, ribosomal protein L19, FtsZ, Sigma 70 and exonuclease VII), or Anaplasmataceae (Tgt and RP-314), and they distinguish these groups from all others. Signatures in DnaA, RP-057, and DNA ligase A are commonly shared by various Rhizobiales, Rhodobacterales, and Caulobacter, suggesting that these groups shared a common ancestor exclusive of other alpha-proteobacteria. A specific relationship between Rhodobacterales and Caulobacter is indicated by a large insert in the Asn-Gln amidotransferase. The Rhizobiales group of species are distinguished from others by a large insert in the Trp-tRNA synthetase. Signature sequences in a number of other proteins (viz. oxoglutarate dehydogenase, succinyl-CoA synthase, LytB, DNA gyrase A, LepA, and Ser-tRNA synthetase) serve to distinguish the Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae families from Bradyrhizobiaceae and Methylobacteriaceae. Based on the distribution patterns of these signatures, it is now possible to logically deduce a model for the branching order among alpha-proteobacteria, which is as follows: Rickettsiales --> Rhodospirillales-Sphingomonadales --> Rhodobacterales-Caulobacterales --> Rhizobiales (Rhizobiaceaea-Brucellaceae-Phyllobacteriaceae, and Bradyrhizobiaceae). The deduced branching order is also consistent with the topologies in the 16 rRNA and other phylogenetic trees. Signature sequences in a number of other proteins provide evidence that alpha-proteobacteria is a late branching taxa within Bacteria, which branched after the delta,epsilon-subdivisions but prior to the beta,gamma-proteobacteria. The shared presence of many of these signatures in the mitochondrial (eukaryotic) homologs also provides evidence of the alpha-proteobacterial ancestry of mitochondria.
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PMID:Protein signatures distinctive of alpha proteobacteria and its subgroups and a model for alpha-proteobacterial evolution. 1598 34


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