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:6.5.1.1 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.
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PMID:DNA helicase IV from HeLa cells. 164 52

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

The Escherichia coli rho 026 mutation that alters the transcription termination protein Rho prevents growth of wild-type bacteriophage T4. Among the consequences of this mutation are delayed and reduced T4 DNA replication. We show that these defects can be explained by defective synthesis of certain T4 replication-recombination proteins. Expression of T4 gene 41 (DNA helicase/primase) is drastically reduced, and expression of T4 genes 43 (DNA polymerase), 30 (DNA ligase), 46 (recombination nuclease), and probably 44 (DNA polymerase-associated ATPase) is reduced to a lesser extent. The compensating T4 mutation goF1 partially restores the synthesis of these proteins and, concomitantly, the synthesis of T4 DNA in the E. coli rho mutant. From analyzing DNA synthesis in wild-type and various multiply mutant T4 strains, we infer that defective or reduced synthesis of these proteins in rho 026-infected cells has several major effects on DNA replication. It impairs lagging-strand synthesis during the primary mode of DNA replication; it delays and depresses recombination-dependent (secondary mode) initiation; and it inhibits the use of tertiary origins. All three T4 genes whose expression is reduced in rho 026 cells and whose upstream sequences are known have a palindrome containing a CUUCGG sequence between the promoter(s) and ribosome-binding site. We speculate that these palindromes might be important for factor-dependent transcription termination-antitermination during normal T4 development. Our results are consistent with previous proposals that the altered Rho factor of rho 026 may cause excessive termination because the transcription complex does not interact normally with a T4 antiterminator encoded by the wild-type goF gene and that the T4 goF1 mutation restores this interaction.
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PMID:Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants. 254 60

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.
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PMID:DNA mismatch correction in a defined system. 266 76

Coordinated DNA synthesis of both strands at the replication fork by a fixed 'replisome' may cause dynamic and topological problems. Based upon known properties of DNA helicase, DNA primase and DNA topoisomerases, and on novel properties of DNA polymerases and DNA ligase, we propose a 'double-loop' model for the replication of eukaryotic DNA that could minimize such problems.
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PMID:A double-loop model for the replication of eukaryotic DNA. 273 71

We have purified a high molecular weight complex (RC-1) from calf thymus nuclei that catalyzes a recombinational repair of double-strand gaps and deletions in DNA by gene conversion as well as cross-over events leading to cointegrant products. These have been detected by polymerase chain reaction analysis using oligonucleotide primer pairs that detect joined sequences originally present on only one or the other of the recombination substrates. RC-1 has an apparent molecular mass of about 550-600 kDa and contains at least five polypeptide chains: molecular masses about 230, 210, 160, 130, and 40 kDa. RC-1 contains a DNA polymerase, identified as DNA polymerase epsilon, that co-purifies with RC-1. A DNA ligase, most likely mammalian DNA ligase III, and a 5'-3' exonuclease also copurify with the RC-1. Most preparations of RC-1 contain low levels of a double-strand endonuclease, 3'-5' exonuclease and single-strand nuclease activities. However, DNA helicase, terminal deoxynucleotidyl transferase, or DNA topoisomerase I and II were not detected in RC-1. The DNA polymerase and DNA ligase in RC-1 can act in concert to repair a multiply gapped DNA to a covalently repaired duplex. The bovine single-strand-binding protein stimulates the formation of the recombination products and the repair reaction mentioned above about 4-fold.
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PMID:A mammalian protein complex that repairs double-strand breaks and deletions by recombination. 839 64

Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.
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PMID:Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II. 864 39

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
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PMID:Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction. 866 52

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.
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PMID:The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex. 879 42

We have examined the stability of long tracts of CAG repeats in yeast mutants defective in enzymes suspected to be involved in lagging strand replication. Alleles of DNA ligase (cdc9-1 and cdc9-2) destabilize CAG tracts in the stable tract orientation, i.e., when CAG serves as the lagging strand template. In this orientation nearly two-thirds of the events recorded in the cdc9-1 mutant were tract expansions. While neither DNA ligase allele significantly increases the frequency of tract-length changes in the unstable orientation, the cdc9-1 mutant produced a significant number of expansions in tracts of this orientation. A mutation in primase (pri2-1) destabilizes tracts in both the stable and the unstable orientations. Mutations in a DNA helicase/deoxyribonuclease (dna2-1) or in two RNase H activities (rnh1Delta and rnh35Delta) do not have a significant effect on CAG repeat tract stability. We interpret our results in terms of the steps of replication that are likely to lead to expansion and to contraction of CAG repeat tracts.
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PMID:The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast. 1092 64


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