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)

It was shown that bacteriophage tf as well as bacteriophages phi p4/40, phi p25/42, phi p23/40 and phi p6/40, which are specific to different P. putida strains, contain the single strand breaks in their DNA. The breaks are localized in one strand of DNA molecules and are repairable with T4 DNA ligase. Bacteriophage tf has no detectable DNA homology with phi p4/40, phi p25/42, phi p23/40 and phi p6/40 bacteriophages. All the phages studied have no relation with other known Pseudomonas phages. Bacteriophages phi p4/40 and phi p25/42 share the extensive DNA homology.
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PMID:[Bacteriophages of Pseudomonas putida containing single-stranded canonical DNA breaks]. 313 62

Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.
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PMID:Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. 1091 87

Pseudomonas aeruginosa encodes two putative DNA ligases: a classical NAD(+)-dependent DNA ligase (LigA) plus an ATP-dependent DNA ligase (LigD). LigD exemplifies a family of bacterial proteins that consist of a ligase domain fused to flanking domains that resemble nucleases and/or polymerases. Here we purify LigD and show that it possesses an intrinsic polymerase function resident within an autonomous C-terminal polymerase domain, LigD-(533-840), that flanks an autonomous DNA ligase domain, LigD-(188-527). Native LigD and the polymerase domain are both monomeric proteins. The polymerase activity is manifest in three ways: (i) non-templated nucleotide addition to a blunt-ended duplex DNA primer; (ii) non-templated addition to a single-stranded DNA primer; and (iii) templated extension of a 5'-tailed duplex DNA primer-template. The divalent cation cofactor requirement for non-templated and templated polymerase activity is satisfied by manganese or cobalt. rNTPs are preferred over dNTPs as substrates for non-templated blunt-end addition, which typically entails the incorporation of only 1 or 2 nucleotides at the primer terminus. Templated dNMP addition to a 5'-tailed substrate is efficient with respect to dNTP utilization; the primer is elongated to the end of the template strand and is then further extended with a non-templated nucleotide. The polymerase activity is abolished by alanine substitution for two aspartates (Asp-669 and Asp-671) within the putative metal-binding site. We speculate that polymerase activity is relevant to LigD function in nonhomologous end-joining.
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PMID:A primer-dependent polymerase function of pseudomonas aeruginosa ATP-dependent DNA ligase (LigD). 1552 14

Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.
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PMID:Novel 3'-ribonuclease and 3'-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D. 1589 97

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3'-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3'-ribonucleotide tract. The phosphodiesterase, which cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus, requires the vicinal 2'-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3'-PO4 to a 3'-OH. Here we show that the PE domain has a 3'-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2'-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3'-phosphatase activity but not for 3' ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3'-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.
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PMID:Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme. 1604 7

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.
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PMID:Genome comparison of Pseudomonas aeruginosa large phages. 1625 35

DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5-A crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP/dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.
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PMID:Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D. 1644 39

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4 degrees C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/microg.
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PMID:Characterization of the bacteriophage PhiKMV DNA ligase. 1652 77

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.
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PMID:Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D. 1654 Apr 77

DNA ligase D (LigD) participates in a mutagenic pathway of nonhomologous end joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase module. The POL domain performs templated and nontemplated primer extension reactions with either dNTP or rNTP substrates. Here we report that Pseudomonas LigD POL is an unfaithful nucleic acid polymerase. Although the degree of infidelity in nucleotide incorporation varies according to the mispair produced, we find that a correctly paired ribonucleotide is added to the DNA primer terminus more rapidly than the corresponding correct deoxyribonucleotide and incorrect nucleotides are added much more rapidly with rNTP substrates than with dNTPs, no matter what the mispair configuration. We find that 3' mispairs are extended by LigD POL, albeit more slowly than 3' paired primer-templates. The magnitude of the rate effect on mismatch extension varies with the identity of the 3' mispair, but it was generally the case that mispaired ends were extended more rapidly with rNTP substrates than with dNTPs. These results lend credence to the suggestion that LigD POL might fill in short 5'-overhangs with ribonucleotides when repairing double strand breaks in quiescent cells. We report that LigD POL can add a deoxynucleotide opposite an abasic lesion in the template strand, albeit slowly. Ribonucleotides are inserted more rapidly at an abasic lesion than are deoxys. LigD POL displays feeble activity in extending a preformed primer terminus opposing an abasic site, but can readily bypass the lesion by slippage of the primer 3' di- or trinucleotide and realignment to the template sequence distal to the abasic site. Covalent benzo[a]pyrene-dG and benzo[c]phenanthrene-dA adducts in the template strand are durable roadblocks to POL elongation. POL can slowly insert a dNMP opposite the adduct, but is impaired in the subsequent extension step.
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PMID:Nucleotide misincorporation, 3'-mismatch extension, and responses to abasic sites and DNA adducts by the polymerase component of bacterial DNA ligase D. 1681 88


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