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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.
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PMID:Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. 815 90

In humans, DNA lesions such as pyrimidine dimers in the template strand of genes transcribed by RNA polymerase II are repaired faster than those in the coding strand and nontranscribed regions of the genome. This phenomenon, referred to as transcription-coupled repair (i) requires active transcription, (ii) does not require the XPC gene product which is essential for general/basal repair reactions, and (iii) requires the CSA and CSB proteins. We have developed an in vitro model system that consists of purified human excision repair factors and a DNA substrate analogous to a transcription bubble terminating at a cyclobutane thymine dimer. In this system the thymine dimer was excised independent of XPC. Furthermore, the thymine dimer in the bubble-containing substrate was removed approximately 3-fold faster by the excision repair nuclease reconstituted with or without XPC, compared with the removal of thymine dimer from a base paired duplex by the entire set of excision nuclease factors. These results provide important insight into the mechanism of transcription-coupled repair in humans.
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PMID:Model for XPC-independent transcription-coupled repair of pyrimidine dimers in humans. 906 8

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells have specific DNA repair defects. We had previously analyzed repair rates of cyclobutane pyrimidine dimers at nucleotide resolution along the human JUN gene in normal fibroblasts and found very efficient repair of sequences near the transcription initiation site but slow repair along the promoter. To investigate sequence-specific repair rate patterns in XP and CS cells, we conducted a similar analysis in XPA, XPB, XPC, XPD, and CSB fibroblasts. XPA cells were almost completely repair-deficient at all sequences analyzed. XPC cells repaired only the transcribed DNA strand beginning at position -20 relative to the transcription start site. Both XBP and XPD cells were deficient in repair of nontranscribed DNA and also very inefficiently repaired the transcribed strand including sequences near the transcription start site. CSB cells exhibited rapid repair near the transcription initiation site but were deficient in repair of sequences encountered by RNA polymerase during elongation (beginning at position +20). Since transcription of the JUN gene was UV-induced in all fibroblast strains, including CSB, the defective repair of the transcribed strand in CSB cannot be explained by a lack of transcription; rather, it appears to be a true DNA repair defect.
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PMID:Sequence-specific and domain-specific DNA repair in xeroderma pigmentosum and Cockayne syndrome cells. 925 97

The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled nucleotide excision repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.
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PMID:Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. 973 59

Some types of damage to cellular DNA have been shown to interfere with the essential transactions of replication and transcription. Not only may the translocation of the polymerase be arrested at the site of the lesion but the bound protein may encumber recognition of the lesion by repair enzymes. In the case of transcription a subpathway of excision repair, termed transcription-coupled repair (TCR) has been shown to operate on lesions in the transcribed strands of expressed genes in bacteria, yeast, mammalian cells and a number of other organisms. Certain genes in mammalian cells (e.g., CSA and CSB) have been uniquely implicated in TCR while others (e.g., XPC-HR23 and XPE) have been shown to operate in the global genomic pathway of nucleotide excision repair, but not in TCR. In order to understand the mechanism of TCR it is important to learn how an RNA polymerase elongation complex interacts with a damaged DNA template. That relationship is explored for different lesions and different RNA polymerase systems in this article.
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PMID:Effect of DNA lesions on transcription elongation. 1021 18

The XPA protein is essential for both of the known modes of nucleotide excision repair (NER) in human cells: transcription-coupled repair (TCR) and global genome repair (GGR). In TCR, this protein is thought to be recruited to lesion sites in DNA at which RNA polymerase II is blocked and in GGR, by direct recognition of damages by repair protein complex containing XPC/HR23B or DNA damage-binding protein. However, details of the recruitment of the XPA protein in vivo are unknown. It was shown earlier that a portion of another NER protein, PCNA, which is completely extractable from non-S-phase mammalian nuclei, becomes insoluble after ultraviolet (UV) light irradiation and cannot be extracted by methanol or buffer containing Triton X-100. In the present study, we have found that UV light irradiation of human or Chinese hamster cells leads to decrease of extractability of the XPA protein by Triton X-100. Maximal insolubilization of the XPA protein is observed 1-4 h after irradiation but it is not detectable by 22 h. This effect is dose-dependent for UV light from 2.5 to 15 J/m(2) and is unaffected by the pre-treatment of cells with sodium butyrate, an inhibitor of histone deacetylation. The UV light-induced insolubilization of the XPA protein was also observed in two lines of Cockayne syndrome complementation group A cells, indicating that the effect is not dependent upon TCR. The results are discussed in relation to possible mechanisms of NER.
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PMID:Reduced extractability of the XPA DNA repair protein in ultraviolet light-irradiated mammalian cells. 1060 36

The multisubunit basal transcription factor IIH (TFIIH) has a dual involvement in nucleotide excision repair (NER) of a variety of DNA lesions, including UV-induced photoproducts, and RNA polymerase II transcription. In both processes, TFIIH is implicated with local DNA unwinding, which is attributed to its helicase subunits XPB and XPD. To further define the role of TFIIH in NER, functional interactions between TFIIH and other DNA repair proteins were analyzed. We show that the TFIIH-associated ATPase activity is stimulated by both XPA and the XPC-HR23B complex. However, while XPA promotes the ATPase activity specifically in the presence of damaged DNA, stimulation by XPC-HR23B is lesion independent. Furthermore, we reveal that TFIIH inhibits the structure-specific endonuclease activities of both XPG and ERCC1-XPF, responsible for the 3' and 5' incision in NER, respectively. The inhibition occurs in the absence of ATP and is reversed upon addition of ATP. These results point toward additional roles for TFIIH and ATP during NER distinct from a requirement for DNA unwinding in the regulation of the endonuclease activities of XPG and ERCC1-XPF.
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PMID:Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF. 1114 Oct 66

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.
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PMID:Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts. 1129 53

To understand the relationship between DNA repair, apoptosis, transcription, and cancer-proneness, we have studied the apoptotic response and the recovery of RNA synthesis following ultraviolet C and ultraviolet B irradiation in nucleotide excision repair deficient diploid fibroblasts from the cancer-prone xeroderma pigmentosum (XP) syndrome patients and the non-cancer-prone trichothiodystrophy (TTD) patients. Analysis of four XPD and four TTD/XPD fibroblast strains presenting different mutations on the XPD gene has shown that XPD cells are more sensitive to ultraviolet-induced apoptosis than TTD/XPD cells, and this response seems to be modulated by the type and the location of the mutation on the XPD gene. Moreover, the other xeroderma pigmentosum fibroblast strains analyzed (groups A and C) are more sensitive to undergo apoptosis after ultraviolet irradiation than normal human fibroblasts, showing that the cancer-proneness of xeroderma pigmentosum patients is not due to a deficiency in the ultraviolet-induced apoptotic response. We have also found that cells from transcription-coupled repair deficient XPA, XPD, TTD/XPD, and Cockayne's syndrome patients undergo apoptosis at lower ultraviolet doses than transcription-coupled repair proficient cells (normal human fibroblasts and XPC), indicating that blockage of RNA polymerase II at unrepaired lesions on the transcribed strand is the trigger. Moreover, XPD and XPA cells are more sensitive to ultraviolet-induced apoptosis than trichothiodystrophy and Cockayne's syndrome fibroblasts, suggesting that both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone on the transcribed strand trigger apoptosis. Finally, we show that apoptosis is directly proportional to the level of inhibition of transcription, which depends on the density of ultraviolet-induced lesions occurring on transcribed sequences.
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PMID:Effects of XPD mutations on ultraviolet-induced apoptosis in relation to skin cancer-proneness in repair-deficient syndromes. 1171 Sep 28

The hepatocarcinogen 2-acetylaminofluorene is one of the most studied experimental carcinogens. We have shown previously that normal rat hepatocytes accumulate the tumour suppressor p53 after exposure to this compound while preneoplastic rat hepatocytes do not. We suggested that the lack of p53 response may confer a growth advantage on preneoplastic hepatocytes and may be an important factor in hepatic tumor promotion by 2-acetylaminofluorene and other genotoxic compounds. Inhibition of RNA polymerase II driven transcription by DNA lesions may constitute one of the mechanisms leading to accumulation of the tumour suppressor p53. We have investigated the accumulation of p53 by structurally different DNA lesions of 2-acetylaminofluorene for which the rate of nucleotide excision repair (NER) and inhibition of transcription are known. Experiments were performed with NER proficient human fibroblasts as well as repair deficient xeroderma pigmentosum group A (XPA) cells, XPC cells [only transcription coupled repair (TCR)] and Cockayne syndrome (CS)B cells [only global genome repair (GGR)]. The cells were exposed to N-acetoxy-acetylaminofluorene (NAAAF) in the presence or absence of paraoxon inducing dG-C8-AAF or dG-C8-AF adducts respectively. Both treatments led to accumulation of p53 in all cells. However, dG-C8-AAF adducts produced greater p53 induction than dG-C8-AF adducts. The percentage p53-positive cells was highest and the threshold for p53 accumulation was lowest in XPA and CSB cells. Our results further demonstrate that both the potency of a lesion to inhibit transcription as well as the restoration of RNA synthesis determines the magnitude of p53 induction.
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PMID:Blockage of transcription as a trigger for p53 accumulation by 2-acetylaminofluorene DNA-adducts. 1288 15


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