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)

Saccharomyces cerevisiae mutants lacking the structure-specific nuclease Rad27 display an enhancement in recombination that increases as sequence length decreases, suggesting that Rad27 preferentially restricts recombination between short sequences. Since wild-type alleles of both RAD27 and its human homologue FEN1 complement the elevated short-sequence recombination (SSR) phenotype of a rad27-null mutant, this function may be conserved from yeast to humans. Furthermore, mutant Rad27 and FEN-1 enzymes with partial flap endonuclease activity but without nick-specific exonuclease activity partially complement the SSR phenotype of the rad27-null mutant. This suggests that the endonuclease activity of Rad27 (FEN-1) plays a role in limiting recombination between short sequences in eukaryotic cells.
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PMID:Novel function of Rad27 (FEN-1) in restricting short-sequence recombination. 1125 84

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.
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PMID:Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes. 1143 21

Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.
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PMID:Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region. 1147 73

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5'-flap strands of the branched DNA structure and possesses 5'-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3'-end of the primer were synthesized in situ by the action of DNA polymerase beta using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5'-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5'- and 3'-ends of the nick or with primer-template systems containing 5'- or 3'-protruding DNA strands was shown. Formation of a structure with the 5'-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3'-end and the apurine/apyrimidine site at the 5'-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.
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PMID:Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs. 1156 52

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

The biochemical and cell cycle-dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease-1 (OsFEN-1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN-1 and increased the flap-endonuclease activity of OsFEN-1 by 2.5-fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN-1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN-1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN-1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10-20 intense spots at leptotene. The number of spots decreased to 1-5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN-1 in mitotic and meiotic cell cycles are discussed.
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PMID:Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease-1 (FEN-1), and their distribution in mitotic and meiotic cell cycles. 1185 10

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.
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PMID:DNA mismatch repair and mutation avoidance pathways. 1192 Jun 79

The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1-/-) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1-/- cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.
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PMID:Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2. 1213 9

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

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.
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PMID:Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1. 1243 4


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