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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multisubunit basal transcription factor IIH (TFIIH) has a dual involvement in nucleotide excision repair (NER) of a variety of DNA lesions, including UV-induced photoproducts, and RNA polymerase II transcription. In both processes, TFIIH is implicated with local DNA unwinding, which is attributed to its helicase subunits XPB and XPD. To further define the role of TFIIH in NER, functional interactions between TFIIH and other DNA repair proteins were analyzed. We show that the TFIIH-associated ATPase activity is stimulated by both XPA and the XPC-HR23B complex. However, while XPA promotes the ATPase activity specifically in the presence of damaged DNA, stimulation by XPC-HR23B is lesion independent. Furthermore, we reveal that TFIIH inhibits the structure-specific endonuclease activities of both XPG and ERCC1-XPF, responsible for the 3' and 5' incision in NER, respectively. The inhibition occurs in the absence of ATP and is reversed upon addition of ATP. These results point toward additional roles for TFIIH and ATP during NER distinct from a requirement for DNA unwinding in the regulation of the endonuclease activities of XPG and ERCC1-XPF.
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PMID:Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF. 1114 Oct 66

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.
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PMID:Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I. 1117 2

Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.
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PMID:RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. 1147 23

The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease. This fork regression may be catalyzed by RecG but is thought to occur even in its absence. Here we test whether RuvAB helicase can also catalyze the unwinding of forked DNA to form Holliday junctions. We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm. If the binding of RuvB is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction. This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer. These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation.
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PMID:Action of RuvAB at replication fork structures. 1155 67

Nucleotide excision repair (NER) removes bulky DNA lesions and is thus crucial for the protection against environmental carcinogens and UV light exposure. Deficiencies in NER cause increased mutation rates and chromosomal aberrations. Current methods for studying NER are mostly based on either quantitation of lesion removal or detection of repair DNA synthesis. Both have their limitations: lesion removal is inaccurate at very short times post-lesion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair from replicative DNA synthesis. To overcome these problems we developed a method for analysis of NER based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabolically labelled with BrdU, exposed to UV-irradiation and the ssDNA transients generated during excision repair are detected using an anti-BrdU antibody. The method allows single-cell microscopic analysis of the distribution of DNA repair sites as well as kinetic analysis of the DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the detection method integrates a number of pre-synthesis nucleotide excision repair stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficient cells.
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PMID:Analysis of nucleotide excision repair by detection of single-stranded DNA transients. 1169 40

F 11782, or 2',3'-bis-pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D-glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin 2-N-methyl glucamine salt, a novel dual catalytic inhibitor of topoisomerases I and II, was identified as a potent inhibitor of nucleotide excision repair (NER) by screening procedures using the in vitro 3D (DNA damage detection) assay. F 11782 was then shown predominantly to inhibit the incision rather than the repair synthesis step, using two new methodologies derived from this 3D assay, effectively ruling out any inhibition of polymerases delta/var epsilon. Moreover, data from two other in vitro assays showed an absence of any effect of F 11782 on: (i) the DNA damage binding of the XPA-RPA complex, and (ii) on SV40 large T-antigen helicase activity. Therefore, the inhibitory activity of F 11782 on NER may involve an inhibition of the ERCC1-XPF or XPG endonuclease activity. Moreover, inhibition of DNA repair by F 11782 was confirmed in human A549 cells by monitoring unscheduled DNA synthesis following mechlorethamine treatment. Such an inhibition provides an explanation for the highly synergistic cytotoxicity observed against cultured A549 lung tumour cells, when F 11782 was combined with cross-linking agents, such as cisplatin or mitomycin C. These results emphasise the unique mode of action of this novel molecule in inhibiting NER and provide a basis for its evaluation in clinical trials in combination with DNA cross-linking agents.
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PMID:Inhibition of nucleotide excision repair and sensitisation of cells to DNA cross-linking anticancer drugs by F 11782, a novel fluorinated epipodophylloid. 1184

Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host. Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus. Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase. Two other compensatory changes of unknown function also occurred. Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion. In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome. The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions.
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PMID:Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. 1186 82

Werner syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability, and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1432-amino acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, overexpressed, and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N(70-240)), exhibits the same level of 3'-5' exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5'-protruding DNA strand endonuclease activity at a single-strand-double-strand DNA junction and within single-stranded DNA, as well as a 3'-5' exonuclease activity on single-stranded DNA. We find hWRN-N(70-240) is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy. Upon addition of DNA substrate, hWRN-N(70-240) forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N(70-240) with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications these results have on our understanding of hWRN's roles in DNA replication and repair are discussed.
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PMID:A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities. 1186 28

A 22.4 kb naturally occurring plasmid pJW566, isolated from L. lactis W56, was found to encode an R/M system named LlaBIII. The LlaBIII R/M system was isolated on a chloramphenicol resistant derivative of plasmid pJW566, resulting in a plasmid pJK1. Subcloning analysis showed that the LlaBIII determinant was located on a 5 kb HindIII-Sph I fragment. The fragment was sequenced. It contained a single open reading frame (ORF), corresponding to a protein of 1584 or 1576 aa. In the deduced amino acid sequence seven helicase motifs characteristic of endonuclease type I and type III and a conserved catalysis motif X in the R subunits of type I R/M systems were located in the N-terminus, followed by four conserved motifs found in DNA N6-adenine methyltransferases. The C-terminus of the deduced amino acid sequence showed no homology to known R/M systems. Therefore, this polypeptide encoded by LlaBIII is a multifunctional protein possessing putative DNA recognition, methylation and restriction activities.
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PMID:[Cloning and structure analysis of a restriction and modification system, LlaBIII from Lactococcus lactis subsp. cremoris W56]. 1191 Jul 61

Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.
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PMID:Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing. 1200 53


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