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 XPF and ERCC1 proteins form a tight complex and function as an endonuclease to incise on the 5'-side of pyrimidine dimers in DNA. Levels of both proteins are extremely low in group F xeroderma pigmentosum (XP-F) cells. We transfected XP-F cells with the plasmids expressing XPF or ERCC1 and examined levels of both proteins in the cells. Although XP-F cells are sensitive to UV and mitomycin C (MMC), cells overexpressing XPF expressed ERCC1 as well and resistance to UV and MMC was restored to the normal level. In contrast, cells overexpressing ERCC1 did not express XPF and were still sensitive to UV and MMC. These results indicate that both the XPF and ERCC1 proteins are required to repair UV- and MMC-induced DNA damage. Even though a high level of ERCC1, which has been presumed to be a catalytic subunit of the endonuclease, is stably present in XP-F cells, ERCC1 protein alone cannot carry out excision repair completely.
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PMID:Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression. 986 90

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.
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PMID:Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours. 1007 55

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 +/- 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.
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PMID:Action of DNA repair endonuclease ERCC1/XPF in living cells. 1032 Mar 75

During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex. To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E. coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit. Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain. Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF. To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding. Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1. We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events. In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation. A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1.
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PMID:Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF. 1041 17

The human XPF protein, an endonuclease subunit essential for DNA excision repair, may also function in homologous recombination. To investigate a possible link between mammalian XPF and recombination that occurs during meiosis, we isolated, characterized, and determined an expression profile for the mouse Xpf gene. The predicted mouse XPF protein, encoded by a 3.4-kb cDNA, contains 917 amino acids and is 86% identical to human XPF. Appreciable similarity also exists between mouse XPF and homologous proteins in budding yeast (Rad1), fission yeast (Rad16), and fruit fly (Mei-9), all of which have dual functions in excision repair and recombination. Sequence analysis of the 38.3-kb Xpf gene, localized to a region in proximal mouse chromosome 16, revealed greater than 72% identity to human XPF in 16 regions. Of these conserved elements, 11 were exons and 5 were noncoding sequence within introns. Xpf transcript and protein levels were specifically elevated in adult mouse testis. Moreover, increased levels of Xpf and Ercc1 mRNAs correlated with meiotic and early postmeiotic spermatogenic cells. These results support a distinct role for the XPF/ERCC1 junction-specific endonuclease during meiosis, most likely in the resolution of heteroduplex intermediates that arise during recombination.
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PMID:Characterization of the mouse Xpf DNA repair gene and differential expression during spermatogenesis. 1064 40

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.
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PMID:DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts. 1071 68

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.
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PMID:Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1. 1075 2

Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.
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PMID:Nucleotide excision repair endonuclease genes in Drosophila melanogaster. 1081 34

The gene MUS81 p6ethyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait. It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals. Mus81p also shares homology with motifs found in the XPF endonuclease superfamily. Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype. However, mus81delta cells were not significantly more sensitive than wild-type to gamma-radiation or double-strand breaks induced by HO endonuclease. Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage. A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments. Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another. Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana. We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway.
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PMID:MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae. 1090 49

Increased platinum-DNA adduct removal has been shown by several DNA repair assays to be associated with cisplatin resistance in the A2780/C-series human ovarian cancer model system. In the present study, we provide further evidence that the resistance phenotype of these cell lines is due, in part, to enhanced nucleotide excision repair (NER). Cisplatin resistance was found to be associated with increased UV resistance. Northern blot analysis revealed that increased expression of ERCC1 was also associated with cisplatin resistance in this panel. Several other NER genes were found to be constitutively overexpressed in the most resistant cell line, C200, as compared with the parental A2780 cells. A plasmid substrate containing a site-specific cisplatin adduct was used to measure the nucleotide excision activity of cell extracts prepared from cisplatin-sensitive and -resistant cells. Using this in vitro assay, extracts prepared from C200 cells exhibited approximately 3-fold more activity than extracts prepared from A2780 cells, similar to the difference in UV sensitivity. Complementation of A2780 extracts with ERCC1-XPF protein resulted in approximately 2-fold increased activity, but had little effect on excision in C200 extracts. Overall, these results support a role for the ERCC1-XPF endonuclease as a determinant of increased NER in this cisplatin resistance model.
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PMID:Increased nucleotide excision repair in cisplatin-resistant ovarian cancer cells: role of ERCC1-XPF. 1100 24


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