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)

Uterine leiomyomas are the most common benign smooth muscle tumors in the myometrium. The expression of redox factor 1 (Ref-1), a DNA repair enzyme and redox-modifying factor, was studied in the myometrium and uterine smooth muscle tumors to investigate the relevance of Ref-1 in the growth regulation of the tumors. Two forms of Ref-1 protein were detected, using three antibodies against different epitopes of Ref-1. The abundance of the large form of Ref-1 was increased in leiomyoma extracts relative to myometrial tissue extracts, and the large form was dominant in cell lines derived from leiomyosarcomas. A single mRNA transcript was detected in the same samples, leading us to hypothesize that the differentially migrating forms are the result of posttranslational modification(s). In vitro incubation of leiomyoma tissue extract lead to a shift from the large form to the small form, and this conversion was inhibited by either protease or phosphatase inhibitors. Finally, the relative abundance of the large form of Ref-1 was found to correlate with proliferating cell nuclear antigen levels, suggesting a correlation with increased proliferation. These results indicate that altered posttranslational modification of Ref-1 is involved in uterine smooth muscle tumorigenesis.
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PMID:Altered post-translational modification of redox factor 1 protein in human uterine smooth muscle tumors. 1216 6

To address the different functions of Pol delta and FEN1 (Rad27) in Okazaki fragment maturation, exonuclease-deficient polymerase Pol delta-01 and Pol delta-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo(-) Pol delta carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol delta-exo(-). When Pol delta-exo(-) encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol delta exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol delta-exo(-) proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo(-) rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo(-) rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonuclease-deficient Pol delta.
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PMID:Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3'-5'-exonuclease activities of Pol delta in the creation of a ligatable nick. 1242 37

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s. When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.
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PMID:Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. 1242 38

The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.
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PMID:A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus. 1253 40

Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase.
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PMID:Plant DNA polymerase lambda, a DNA repair enzyme that functions in plant meristematic and meiotic tissues. 1520 45

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).
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PMID:Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells. 1547 84

DNA polymerase (Pol) lambda is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol lambda has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol lambda has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol lambda. Pol lambda is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol lambda was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol lambda was decreased by its interaction with PCNA. Finally, Pol lambda is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle.
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PMID:Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen. 1617 46

DNA ligase is an essential enzyme for all organisms and catalyzes a nick-joining reaction in the final step of the DNA replication, repair, and recombination processes. Herein, we show the physical and functional interaction between DNA ligase and proliferating cell nuclear antigen (PCNA) from the hyperthermophilic Euryarchaea Pyrococcus furiosus. The stimulatory effect of P. furiosus PCNA on the enzyme activity of P. furiosus DNA ligase was observed not at low ionic strength, but at a high salt concentration, at which a DNA ligase alone cannot bind to a nicked DNA substrate. On the basis of mutational analyses, we identified the amino acid residues that are critical for PCNA binding in a loop structure located in the N-terminal DNA-binding domain of P. furiosus DNA ligase. We propose that the pentapeptide motif QKSFF is involved in the PCNA-interacting motifs, in which Gln and the first Phe are especially important for stable binding with PCNA.
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PMID:Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen. 1682 13

DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate multistep DNA transactions. We have determined structures of Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.
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PMID:A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA. 1705 61

Three-dimensional structures of DNA N-glycosylases and N-glycosylase/apyrimidine/apurine (AP)-lyase enzymes and other critical components of base excision repair (BER) machinery including structure-specific nuclease, repair polymerase, DNA ligase, and PCNA tethering complexes reveal the overall unity of the simple cut and patch process of DNA repair for damaged bases. In general, the damage-specific excision is initiated by structurally-variable DNA glycosylases targeted to distinct base lesions. This committed excision step is followed by a subsequent damage-general processing of the resulting abasic sites and 3' termini, the insertion of the correct base by a repair DNA polymerase, and finally sealing the nicked backbone by DNA ligase. However, recent structures of protein-DNA and protein-protein complexes and other BER machinery are providing a more in-depth look into the intricate functional diversity and complexity of maintaining genomic integrity despite very high levels of constant DNA base damage from endogenous as well as environmental agents. Here we focus on key discoveries concerning BER structural biology that speak to better understanding the damage recognition, reaction mechanisms, conformational controls, coordinated handoffs, and biological activities including links to cancer. As the three-dimensional crystal and NMR structures for the protein and DNA complexes of all major components of the BER system have now been determined, we provide here a relatively complete description of the key complexes starting from DNA base damage detection and excision to the final ligation process. As our understanding of BER structural biology and the molecular basis for cancer improve, we predict that there will be multiple links joining BER enzyme mutations and cancer predispositions, such as now seen for MYH. Currently, structural results are realizing the promise that high-resolution structures provide detailed insights into how these BER proteins function to specifically recognize, remove, and repair DNA base damage without the release of toxic and mutagenic intermediates.
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PMID:The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair. 1720 22


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