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)

A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1 complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with a "bubble" structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication protein A when the DNA contains UV damage.
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PMID:Purification and characterization of the XPF-ERCC1 complex of human DNA repair excision nuclease. 755 82

Human cells from patients suffering with xeroderma pigmentosum (XP) characterized by extreme sensitivity to UV light and a high incidence of skin tumors fall into seven complementation groups, XPA to XPG, and are lacking a functional helicase, endonuclease, or lesion-recognizing protein involved in the initial steps during nucleotide excision repair (NER); a number of proteins involved in DNA repair are termed XPA to XPG depending on which one is defective in a particular complementation group of XP and include: (i) proteins involved in the recognition of (6-4) photoproducts (XPE) and of a broad range of lesions such as pyrimidine dimers (XPA); (ii) proteins that are DNA helicases and integral parts of the general transcription factor TFIIH functioning in both transcription and repair (XPB, XPD); (iii) endonucleases that perform the two incisions, the XPG incising six nucleotides (nt) to the 3' side from a photodimer and the ERCC1-XPF protein complex incising 22 nt to the 5' side of the lesion; and (iv) single-strand DNA-binding proteins (XPC). The ERCC6 helicase is largely responsible for coupling transcription to repair whereas XPC seems to be responsible for the repair of the inactive parts of the genome as well as for the repair of the nontranscribed strand in active genes. p53 recognizes insertion/deletion mismatches as well as free ends of DNA produced by ionizing radiation to arrest the cell cycle. Most of the human DNA repair proteins have their counterparts in both budding and fission yeasts and some of them also in E. coli evoking an evolutionary conservation of DNA repair pathways. Accumulation of mutations within repair genes in single cells followed by their escape from the immune surveillance and in clonal expansion may greatly contribute to the appearance and development of human cancers.
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PMID:Xeroderma pigmentosum and molecular cloning of DNA repair genes. 868 16

Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients. The XPF protein was purified from mammalian cells in a tight complex with ERCC1. This complex is a structure-specific endonuclease responsible for the 5' incision during repair. These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.
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PMID:Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease. 879 27

The repair-deficient mutant rodent cell lines UV20 and UV41, which are defective in the ERCC1/ERCC4[XPF]-mediated 5'-endonuclease activity, are unusually sensitive to gamma-irradiation under hypoxic (but not oxic) conditions. Because this 5'-endonuclease appears to be involved in two distinct (but overlapping) DNA-repair pathways-the nucleotide excision repair pathway and the recombination-dependent pathway for the removal of DNA interstrand cross-links-it is unclear which of these defective activities is responsible for the hypoxic radiosensitivity of UV20 and UV41 cells. Accordingly, we have extended these measurements to the UV5 and UV24 lines which carry mutations in the ERCC2[XPD] and ERCC3[XPB] genes, respectively; both of these genes encode DNA helicases. These two mutants display a sensitivity to ultraviolet light that is similar to that of UV20 and UV41 cells, reflecting their defect in the incision step of the nucleotide excision repair pathway. However, neither UV5 nor UV24 cells are especially cross-sensitive to agents that produce DNA interstrand cross-links, suggesting that the ERCC2 and ERCC3 activities are not crucial for the repair of these lesions. We show that neither UV5 nor UV24 cells exhibit the unusual hypoxic radiosensitivity that characterizes UV20 and UV41 cells. Based on these data and on a comparison of the patterns of cross-sensitivity of these various mutants to other DNA-damaging agents, we conclude that the increased hypoxic radiosensitivity observed in the UV20 and UV41 mutants is due to a defect in the ERCC1/ERCC4-dependent pathway for the repair of DNA cross-links and not in the nucleotide excision repair pathway. The evidence suggests that this sensitivity may be mediated by some type of radiation-induced cross-links, possibly DNA-protein cross-links.
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PMID:The importance of the ERCC1/ERCC4[XPF] complex for hypoxic-cell radioresistance does not appear to derive from its participation in the nucleotide excision repair pathway. 896 Jan 33

The human XPF-ERCC1 protein complex is one of several factors known to be required for general nucleotide excision repair. Genetic data indicate that both proteins of this complex are necessary for the repair of interstrand cross-links, perhaps via recombination. To determine whether XPF-ERCC1 completes a set of six proteins that are sufficient to carry out excision repair, the human XPF and ERCC1 cDNAs were coexpressed in Sf21 insect cells from a baculovirus vector. The purified complex contained the anticipated 5' junction-specific endonuclease activity that is stimulated through a direct interaction between XPF and replication protein A (RPA). The recombinant complex also complemented extracts of XP-F cells and Chinese hamster ovary mutants assigned to complementation groups 1, 4, and 11. Furthermore, reconstitution of the human excision nuclease was observed with a mixture of five repair factors (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1, thus verifying that no additional protein factors are needed for the specific dual incisions characteristic of human excision repair.
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PMID:Reconstitution of human excision nuclease with recombinant XPF-ERCC1 complex. 901 42

XPG is a member of the FEN-1 structure-specific endonuclease family. It has 3'-junction cutting activity on bubble substrates and makes the 3'-incision in the human dual incision (excision nuclease) repair system. To investigate the precise role of XPG in nucleotide excision repair, we mutagenized two amino acid residues thought to be involved in DNA binding and catalysis, overproduced the mutant proteins using a baculovirus/insect cell system, and purified and characterized the mutant proteins. The mutation D77A had a modest effect on junction cutting and excision activity and gave rise to uncoupled 5'-incision by mammalian cell-free extracts. The D812A mutation completely abolished the junction cutting and 3'-incision activities of XPG, but the excision nuclease reconstituted with XPG (D812A) carried out normal 5'-incision at the 23rd-24th phosphodiester bonds 5' to a (6-4) photoproduct without producing any 3'-incision. It is concluded that Asp-812 is an active site residue of XPG and that in addition to making the 3'-incision, the physical presence of XPG in the protein-DNA complex is required non-catalytically for subsequent 5'-incision by XPF-ERCC1.
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PMID:The non-catalytic function of XPG protein during dual incision in human nucleotide excision repair. 918 7

We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1). Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication. Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination. We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells. The reaction gave rise to functional a sequences supporting the formation of defective viruses. It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae. In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells. Our results suggest that homologous recombination may be involved in the circularization of viral genomes. Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.
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PMID:Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4. 926 9

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.
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PMID:DNA structural elements required for ERCC1-XPF endonuclease activity. 952 76

A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32-37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene.
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PMID:Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair. 956 Apr 35

Xeroderma pigmentosum (XP) complementation group F was first reported in Japan and most XP-F patients reported to date are Japanese. The clinical features of XP-F patients are rather mild, including late onset of skin cancer. Recently a cDNA that corrects the repair deficiency of cultured XP-F cells was isolated. The XPF protein forms a tight complex with ERCC1 and this complex functions as a structure-specific endonuclease responsible for the 5' incision during DNA excision repair. Here we have identified XPF mRNA mutations and examined levels of the mRNA and protein expression in seven primary cell strains from Japanese XP-F patients. The XP-F cell strains were classified into three types in terms of the effect of the mutation on the predicted protein; (i) XPF proteins with amino acid substitutions; (ii) amino acid substituted and truncated XPF proteins; and (iii) truncated XPF protein only. A normal level of expression of XPF mRNA was observed in XP-F cells but XPF protein was extremely low. These results indicate that the detected mutations lead to unstable XPF protein, resulting in a decrease in formation of the ERCC1-XPF endonuclease complex. Slow excision repair of UV-induced DNA damage due to low residual endonuclease activity provides a plausible explanation for the typical mild phenotype of XP-F patients.
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PMID:Characterization of molecular defects in xeroderma pigmentosum group F in relation to its clinically mild symptoms. 958 Jun 60


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