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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.
Mol Cell Biol 2001 Apr
PMID:Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome. 1125 78

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.
Mol Cell 2001 Jul
PMID:Sequential assembly of the nucleotide excision repair factors in vivo. 1151 74

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.
Prog Nucleic Acid Res Mol Biol 2001
PMID:Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells. 1155 93

Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damage-containing oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5' to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.
Mol Cell Biol 2002 Aug
PMID:Molecular anatomy of the human excision nuclease assembled at sites of DNA damage. 1213 3

Mutation in the CSB gene results in the human Cockayne's syndrome (CS). Here, we provide evidence that CSB is found not only in the nucleoplasm but also in the nucleolus within a complex (CSB IP/150) that contains RNA pol I, TFIIH, and XPG and promotes efficient rRNA synthesis. CSB is active in in vitro RNA pol I transcription and restores rRNA synthesis when transfected in CSB-deficient cells. We also show that mutations in CSB, as well as in XPB and XPD genes, all of which confer CS, disturb the RNA pol I/TFIIH interaction within the CSB IP/150. In addition to revealing an unanticipated function for CSB in rRNA synthesis, we show that the fragility of this complex could be one factor contributing to the CS phenotype.
Mol Cell 2002 Oct
PMID:CSB is a component of RNA pol I transcription. 1241 26

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.
Plant Mol Biol 2003 Jan
PMID:OsSEND-1: a new RAD2 nuclease family member in higher plants. 1260 91

Archaea share many similarities with eukarya in their information processing pathways and have proven to be a useful model for studies of DNA replication and transcription, but DNA repair pathways are not well understood in archaea. Nucleotide Excision Repair (NER) deals with many bulky DNA lesions and involves over 30 proteins in eukarya. Archaeal NER has not been characterized biochemically, but homologues of the human repair nucleases XPF and XPG have been identified by homology searches. Crenarchaeal XPF proteins have a simplified domain structure, consisting of the C-terminal nuclease domain conserved in XPF and Mus81 but lacking the N-terminal 'helicase' domain that is found in eukaryal and euryarchaeal sequences. Unexpectedly, Sulfolobus XPF is only active in the presence of the sliding clamp PCNA, which is a heterotrimer in this organism. Interactions with two of the three subunits of PCNA are mediated via a C-terminal interaction motif. The PCNA-XPF complex acts as a structure-specific nuclease on a similar range of DNA flap, bubble and junction substrates as the human protein, suggesting a fundamental conservation through billions of years of evolution.
Mol Microbiol 2003 Apr
PMID:An archaeal XPF repair endonuclease dependent on a heterotrimeric PCNA. 1267 97

We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.
Mol Genet Genomics 2003 Aug
PMID:Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae. 1282 2

Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5' to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.
Mol Cell Biol 2004 Feb
PMID:Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF. 1472 65

Cells of budding yeast give rise to mother and daughter cells, which differ in that only mother cells express the HO endonuclease gene and are thereby able to switch mating types. In this study, we identified the MKT1 gene as a positive regulator of HO expression. The MKT1 gene encodes a protein with two domains, XPG-N and XPG-I, which are conserved among a family of nucleases, including human XPG endonuclease. Loss of MKT1 had little effect on HO mRNA levels but resulted in decreased protein levels. This decrease was dependent on the 3' untranslated region of the HO transcript. We screened for proteins that associate with Mkt1 and isolated Pbp1, a protein that is known to associate with Pab1, a poly(A)-binding protein. Loss of PBP1 resembles an mkt1 Delta deletion, causing decreased expression of HO at the posttranscriptional level. Mkt1 and Pbp1 cosedimented with polysomes in sucrose gradients, with Mkt1 distribution in the polysomes dependent on Pbp1, but not vice versa. These observations suggest that a complex of Mkt1 and Pbp1 regulates the translation of HO mRNA.
Mol Cell Biol 2004 May
PMID:Posttranscriptional regulation of HO expression by the Mkt1-Pbp1 complex. 1508 63


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