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)

Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.
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PMID:Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. 1159 21

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

Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.
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PMID:Biochemical characterization of the WRN-FEN-1 functional interaction. 1235 23

Mus81 is a highly conserved substrate specific endonuclease. Human Mus81 cleaves Holliday junctions, replication forks, and 3' flap substrates in vitro, suggesting a number of possible in vivo functions. We show here that the abundance of human Mus81 peaks in S-phase and remains high in cells that have completed DNA replication and that Mus81 is a predominantly nuclear protein, with super accumulation in nucleoli. Two RecQ related DNA helicases BLM and WRN that are required for recombination repair in human cells colocalize with Mus81 in nucleoli. However, the nucleolar retention of Mus81 is not dependent on the presence of BLM or WRN, or on ongoing transcription. Mus81 is recruited to localized regions of UV damage in S-phase cells, but not in cells that are blocked from replicating DNA or that have completed replication. The retention of human Mus81 at regions of UV-induced damage specifically in S-phase cells suggest that the enzyme is recruited to the sites at which replication forks encounter damaged DNA. The nucleolar concentration of Mus81 suggests that it is required to repair problems that arise most frequently in the highly repetitive nucleolar DNA. Together these data support a role for Mus81 in recombination repair in higher eukaryotes.
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PMID:Mus81 endonuclease localizes to nucleoli and to regions of DNA damage in human S-phase cells. 1463 71

Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase-endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN-FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.
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PMID:In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication. 1528 7

Flap endonuclease-1 (FEN-1) is a structure-specific nuclease best known for its involvement in RNA primer removal and long-patch base excision repair. This enzyme is known to possess 5'-flap endo- (FEN) and 5'-3' exo- (EXO) nuclease activities. Recently, FEN-1 has been reported to also possess a gap endonuclease (GEN) activity, which is possibly involved in apoptotic DNA fragmentation and the resolution of stalled DNA replication forks. In the current study, we compare the kinetics of these activities to shed light on the aspects of DNA structure and FEN-1 DNA-binding elements that affect substrate cleavage. By using DNA binding deficient mutants of FEN-1, we determine that the GEN activity is analogous to FEN activity in that the single-stranded DNA region of DNA substrates interacts with the clamp region of FEN-1. In addition, we show that the C-terminal extension of human FEN-1 likely interacts with the downstream duplex portion of all substrates. Taken together, a substrate-binding model that explains how FEN-1, which has a single active center, can have seemingly different activities is proposed. Furthermore, based on the evidence that GEN activity in complex with WRN protein cleaves hairpin and internal loop substrates, we suggest that the GEN activity may prevent repeat expansions and duplication mutations.
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PMID:The DNA-protein interaction modes of FEN-1 with gap substrates and their implication in preventing duplication mutations. 1658 3

Cells repair most double-strand breaks (DSBs) that arise during replication or by environmental insults through homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination machinery-such as Bloom and Werner syndrome proteins-have been resolved. We have taken a novel approach to examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites and visualized in live cells expressing green fluorescent protein-DNA binding protein chimeras. Following induction of an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine the relative positions of the DSB and the template locus. We found a significant increase in persistent associations between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts with a template during the repair process, suggesting that repair could involve interactions with multiple templates, potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these results demonstrate that mechanistic insights into recombination not accessible from studies of populations of cells emerge from observations of individual cells.
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PMID:The dynamics of homologous pairing during mating type interconversion in budding yeast. 1678 29

The progeroid Werner's syndrome (WS) represents the best current model of human aging. It is caused by loss of the WRN helicase/exonuclease, resulting in high levels of replication fork stalling and genomic instability. Current models suggest that characteristic WS phenotypes of poor S phase progression, low proliferative capacity, and drug hypersensitivity are the result of accumulation of alternative DNA structures at stalled or collapsed forks during DNA replication, and Holliday junction resolution has been shown to enhance survival of cis-platin-treated WS cells. Here, we present a direct test of the hypothesis that the replication/repair defect in unstressed WS cells is the result of an inability to resolve recombination intermediates. We have created isogenic WS cell lines expressing a nuclear-targeted bacterial Holliday junction endonuclease, RusA, and show that Holliday junction resolution by RusA restores DNA replication capacity in primary WS fibroblasts and enhances their proliferation. Furthermore, RusA expression rescues WS fibroblast hypersensitivity to replication fork blocking agents camptothecin and 4NQO, suggesting that the hypersensitivity is caused by inappropriate recombination at DNA structures formed when the replication fork arrests or collapses at 4NQO- or camptothecin-induced lesions. This work is the first to demonstrate that Holliday junction accumulation in primary Werner syndrome fibroblasts results in their poor proliferative capacity, and to rescue WS hypersensitivity to camptothecin and 4NQO by Holliday junction resolution.
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PMID:Correction of proliferation and drug sensitivity defects in the progeroid Werner's Syndrome by Holliday junction resolution. 1737 50

Flap endonuclease-1 (FEN1) is a structure specific endonuclease. The natural substrates of FEN1 are 5'-flap structures formed by three DNA chains one of them has unannealed flapped 5'-end (flap). Flap structures are the intermediates of different processes of DNA metabolism, such as DNA recombination, Okazaki fragment maturation during replication of lagging strand, as well as strand displacement DNA synthesis in base excision repair. FEN1 also possesses 5'-exonuclease activity and newly discovered gap endonuclease activity. FEN1 is known to interact physically and functionally with a number of DNA replication and repair proteins such as the proliferating cell nuclear antigen, helicase/nuclease Dna2, WRN and BLM proteins, replication protein A, apurinic/apyrimidinic endonuclease 1, DNA polymerase beta, poly(ADP-riboso) polymerase 1, high mobility group protein 1, integrase of human immunodeficiency virus, transcription coactivator p300, chromatin proteins, cyclin-dependent kinases (Cdk1, Cdk2, Cyclin A). FEN1 activity is significant for maintaining the integrity of repeat sequences in genome. Recent data suppose the correlation between the abnormality of hFEN1 activity and arising/progression of neurodegenerative and cancer diseases. FEN1 has the dramatic effect on cell growth and development thereby attracting the interest to this enzyme.
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PMID:[Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells]. 1870 99

Progressive telomere shortening eventually results in chromosome fusions and genome instability as the cell's ability to distinguish chromosome ends from DNA double-strand breaks is compromised. In fission yeast, such events frequently produce stable survivors with all circular chromosomes. To shed light on the repair pathways that mediate chromosome end fusions and generate circular chromosomes, we have examined a diverse array of DNA repair factors. We show that telomere attrition-induced chromosome fusions are dependent on the fission yeast homologs of Rad52, the ERCC1/XPF endonuclease, the single-stranded DNA-binding protein RPA, and the Srs2 and Werner/Bloom helicases, but not Ku and ligase 4. Consistent with a recombinational mechanism of single-strand annealing, cloned junctions map to four of five homology regions in subtelomeric DNA. A comparison with telomere uncapping caused by the absence of the double-stranded telomere-binding protein Taz1 demonstrates that the circumstances and cause of telomere dysfunction profoundly affect which DNA repair pathway is engaged.
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PMID:Chromosome fusions following telomere loss are mediated by single-strand annealing. 1872 73


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