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Journal articles on the topic 'Transcription coupled repair factors (TCR)'
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1
Guirouilh-Barbat, Josée, Christophe Redon, and Yves Pommier. "Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex." Molecular Biology of the Cell 19, no. 9 (September 2008): 3969–81. http://dx.doi.org/10.1091/mbc.e08-02-0215.
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The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as γ-H2AX foci that colocalize with 53BP1, Mre11, Ser1981-pATM, and Thr68-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK–dependent γ-H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK-γ-H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that γ-H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment.
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Li, Shisheng, Baojin Ding, Runqiang Chen, Christine Ruggiero, and Xuefeng Chen. "Evidence that the Transcription Elongation Function of Rpb9 Is Involved in Transcription-Coupled DNA Repair in Saccharomyces cerevisiae." Molecular and Cellular Biology 26, no. 24 (October 9, 2006): 9430–41. http://dx.doi.org/10.1128/mcb.01656-06.
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ABSTRACT Rpb9, a small nonessential subunit of RNA polymerase II, has been shown to have multiple transcription-related functions in Saccharomyces cerevisiae. These functions include promoting transcription elongation and mediating a subpathway of transcription-coupled repair (TCR) that is independent of Rad26, the homologue of human Cockayne syndrome complementation group B protein. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. Here we show that the Zn1 and linker domains are essential, whereas the Zn2 domain is almost dispensable, for both transcription elongation and TCR functions. Impairment of transcription elongation, which does not dramatically compromise Rad26-mediated TCR, completely abolishes Rpb9-mediated TCR. Furthermore, Rpb9 appears to be dispensable for TCR if its transcription elongation function is compensated for by removing a transcription repression/elongation factor. Our data suggest that the transcription elongation function of Rpb9 is involved in TCR.
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Bucheli, Miriam, Lori Lommel, and Kevin Sweder. "The Defect in Transcription-Coupled Repair Displayed by a Saccharomyces cerevisiae rad26 Mutant Is Dependent on Carbon Source and Is Not Associated With a Lack of Transcription." Genetics 158, no. 3 (July 1, 2001): 989–97. http://dx.doi.org/10.1093/genetics/158.3.989.
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Abstract Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts. Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR). TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators. SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources. The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions. We speculate that another factor(s) can substitute for Rad26 under particular growth conditions. We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source. Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription.
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Desai, Shyamal D., Hui Zhang, Alexandra Rodriguez-Bauman, Jin-Ming Yang, Xiaohua Wu, Murugesan K. Gounder, Eric H. Rubin, and Leroy F. Liu. "Transcription-Dependent Degradation of Topoisomerase I-DNA Covalent Complexes." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2341–50. http://dx.doi.org/10.1128/mcb.23.7.2341-2350.2003.
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ABSTRACT Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR.
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Atanassov, Boyko, Aneliya Velkova, Emil Mladenov, Boyka Anachkova, and George Russev. "Comparison of the Global Genomic and Transcription-Coupled Repair Rates of Different Lesions in Human Cells." Zeitschrift für Naturforschung C 59, no. 5-6 (June 1, 2004): 445–53. http://dx.doi.org/10.1515/znc-2004-5-628.
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There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences. There are data that GGR rates depend on the chemical nature of the lesions in a manner that the lesions inflicting larger distortion on the DNA double helix are repaired at higher rate. It is not known whether the TCR repair rates depend on the type of lesions and in what way. To address this question human cells were transfected with pEGFP and pEYFP plasmids treated with UV light, cis-diamminedichloroplatinum(II) (cisplatin) and angelicin and 24 h later the restored fluorescence was measured and used to calculate the respective NER rates. In a parallel series of experiments the same plasmids were incubated in repair-competent protein extracts to determine GGR rates in the absence of transcription. From the two sets of data, the TCR rates were calculated. We found out that cisplatin, UV light and angelicin lesions were repaired by GGR with different efficiency, which corresponded to the degree of DNA helix distortion induced by these agents. On the other hand the three lesions were repaired by TCR at very similar rates which showed that TCR efficiency was not directly connected with the chemical nature of the lesions.
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Nouspikel, Thierry P., Nevila Hyka-Nouspikel, and Philip C. Hanawalt. "Transcription Domain-Associated Repair in Human Cells." Molecular and Cellular Biology 26, no. 23 (October 2, 2006): 8722–30. http://dx.doi.org/10.1128/mcb.01263-06.
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ABSTRACT Nucleotide excision repair (NER), which is arguably the most versatile DNA repair system, is strongly attenuated in human cells of the monocytic lineage when they differentiate into macrophages. Within active genes, however, both DNA strands continue to be proficiently repaired. The proficient repair of the nontranscribed strand cannot be explained by the dedicated subpathway of transcription-coupled repair (TCR), which is targeted to the transcribed strand in expressed genes. We now report that the previously termed differentiation-associated repair (DAR) depends upon transcription, but not simply upon RNA polymerase II (RNAPII) encountering a lesion: proficient repair of both DNA strands can occur in a part of a gene that the polymerase never reaches, and even if the translocation of RNAPII is blocked with transcription inhibitors. This suggests that DAR may be a subset of global NER, restricted to the subnuclear compartments or chromatin domains within which transcription occurs. Downregulation of selected NER genes with small interfering RNA has confirmed that DAR relies upon the same genes as global genome repair, rather than upon TCR-specific genes. Our findings support the general view that the genomic domains within which transcription is active are more accessible than the bulk of the genome to the recognition and repair of lesions through the global pathway and that TCR is superimposed upon that pathway of NER.
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van den Boom, Vincent, Elisabetta Citterio, Deborah Hoogstraten, Angelika Zotter, Jean-Marc Egly, Wiggert A. van Cappellen, Jan H. J. Hoeijmakers, Adriaan B. Houtsmuller, and Wim Vermeulen. "DNA damage stabilizes interaction of CSB with the transcription elongation machinery." Journal of Cell Biology 166, no. 1 (June 28, 2004): 27–36. http://dx.doi.org/10.1083/jcb.200401056.
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The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.
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D'Errico, Mariarosaria, Massimo Teson, Angelo Calcagnile, Tiziana Nardo, Naomi De Luca, Chiara Lazzari, Silvia Soddu, Giovanna Zambruno, Miria Stefanini, and Eugenia Dogliotti. "Differential Role of Transcription-Coupled Repair in UVB–Induced Response of Human Fibroblasts and Keratinocytes." Cancer Research 65, no. 2 (January 15, 2005): 432–38. http://dx.doi.org/10.1158/0008-5472.432.65.2.
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Abstract Most solar radiation–induced skin cancers arise in keratinocytes. In the human epidermis, protection against cancer is thought to be mediated mainly by nucleotide excision repair (NER) of UVB-induced cyclobutane pyrimidine dimers, and by elimination of the damaged cells by apoptosis. NER consists of two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). Here, we investigate the impact of defects in NER subpathways on the cellular response to UVB-induced damage by comparing primary human keratinocytes and fibroblasts from normal, XP-C (GGR-defective), and CS-A (TCR-defective) individuals. We show that human keratinocytes are more resistant to UVB killing than fibroblasts and present higher levels of UVB-induced DNA repair synthesis due to a more efficient GGR. The CS-A defect is associated with a strong apoptotic response in fibroblasts but not in keratinocytes. Following an UVB dose of 1,000 J/m2, no p53-mediated transactivation of mdm2 is observed in CS-A fibroblasts, whereas the p53-mdm2 circuit is fully activated in CS-A keratinocytes. Thus, in fibroblasts, the signal for apoptosis originates from DNA photoproducts in the transcribed strand of active genes, whereas in keratinocytes, it is largely TCR-independent. This study shows that the response to UVB radiation is cell type–specific in humans and provides the first evidence that a deficiency in TCR has a different impact depending on the cell type. These findings have important implications for the mechanism of skin cancer protection after UVB damage and may explain the lack of skin cancer in patients with Cockayne syndrome.
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Maddukuri, Leena, Dominika Dudzińska, and Barbara Tudek. "Bacterial DNA repair genes and their eukaryotic homologues: 4. The role of nucleotide excision DNA repair (NER) system in mammalian cells." Acta Biochimica Polonica 54, no. 3 (September 23, 2007): 469–82. http://dx.doi.org/10.18388/abp.2007_3222.
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The eukaryotic cell encounters more than one million various kinds of DNA lesions per day. The nucleotide excision repair (NER) pathway is one of the most important repair mechanisms that removes a wide spectrum of different DNA lesions. NER operates through two sub pathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR repairs the DNA damage throughout the entire genome and is initiated by the HR23B/XPC complex, while the CSB protein-governed TCR process removes DNA lesions from the actively transcribed strand. The sequence of events and the role of particular NER proteins are currently being extensively discussed. NER proteins also participate in other cellular processes like replication, transcription, chromatin maintenance and protein turnover. Defects in NER underlay severe genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD).
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Chen, Xuefeng, Christine Ruggiero, and Shisheng Li. "Yeast Rpb9 Plays an Important Role in Ubiquitylation and Degradation of Rpb1 in Response to UV-Induced DNA Damage." Molecular and Cellular Biology 27, no. 13 (April 23, 2007): 4617–25. http://dx.doi.org/10.1128/mcb.00404-07.
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ABSTRACT Rpb9, a nonessential subunit of RNA polymerase II (Pol II), has multiple transcription-related functions in Saccharomyces cerevisiae, including transcription elongation and transcription-coupled repair (TCR). Here we show that, in response to UV radiation, Rpb9 also functions in promoting ubiquitylation and degradation of Rpb1, the largest subunit of Pol II. This function of Rpb9 is not affected by any pathways of nucleotide excision repair, including TCR mediated by Rpb9 itself and by Rad26. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. The Zn2 domain, which is dispensable for transcription elongation and TCR functions, is essential for Rpb9 to promote Rpb1 degradation, whereas the Zn1 and linker domains, which are essential for transcription elongation and TCR functions, play a subsidiary role in Rpb1 degradation. Coimmunoprecipitation analysis suggests that almost the full length of Rpb9 is required for a strong interaction with the core Pol II: deletion of the Zn2 domain causes dramatically weakened interaction, whereas deletion of Zn1 and the linker resulted in undetectable interaction. Furthermore, we show that Rpb1, rather than the whole Pol II complex, is degraded in response to UV radiation and that the degradation is primarily mediated by the 26S proteasome.
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Guintini, Laetitia, Audrey Paillé, Marco Graf, Brian Luke, Raymund J. Wellinger, and Antonio Conconi. "Transcription of ncRNAs promotes repair of UV induced DNA lesions in Saccharomyces cerevisiae subtelomeres." PLOS Genetics 18, no. 4 (April 29, 2022): e1010167. http://dx.doi.org/10.1371/journal.pgen.1010167.
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Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y’, and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.
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Liu, Lili, and Andrew J. Rainbow. "Pre-UV-Treatment of Cells Results in Enhanced Host Cell Reactivation of a UV Damaged Reporter Gene in CHO-AA8 Chinese Hamster Ovary Cells but Not in Transcription-Coupled Repair Deficient CHO-UV61 Cells." Bioscience Reports 24, no. 6 (December 1, 2004): 559–76. http://dx.doi.org/10.1007/s10540-005-2792-x.
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We have used a non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the β-galactosidase reporter gene, to examine both constitutive and inducible repair of UV-damaged DNA in repair proficient CHO-AA8 Chinese hamster ovary cells and in mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of nucleotide excision repair. Host cell reactivation (HCR) of β-galactosidase activity for UV-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UV-damaged reporter gene in untreated cells utilizes TCR. Prior UV-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UV-damaged reporter gene in CHO-AA8 cells but not in TCR deficient CHO-UV61 cells. These results suggest the presence of an inducible DNA pathway in CHO cells that results from an enhancement of TCR or a mechanism that involves the TCR pathway.
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Fei, Jia, and Junjie Chen. "KIAA1530 Protein Is Recruited by Cockayne Syndrome Complementation Group Protein A (CSA) to Participate in Transcription-coupled Repair (TCR)." Journal of Biological Chemistry 287, no. 42 (August 17, 2012): 35118–26. http://dx.doi.org/10.1074/jbc.m112.398131.
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Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UVsS syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UVsS syndrome.
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Sweder, Kevin S., Richard A. Verhage, David J. Crowley, Gray F. Crouse, Jaap Brouwer, and Philip C. Hanawalt. "Mismatch Repair Mutants in Yeast Are Not Defective in Transcription-Coupled DNA Repair of UV-Induced DNA Damage." Genetics 143, no. 3 (July 1, 1996): 1127–35. http://dx.doi.org/10.1093/genetics/143.3.1127.
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Abstract Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively. Other factors probably are also uniquely involved in transcription-repair coupling. Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes. We examined removal of UV-induced DNA damage in yeast strains mutated in mismatch repair genes in an effort to confirm a defect in transcription-coupled repair in this system. In addition, we determined the contribution of the mismatch repair gene MSH2 to transcription-coupled repair in the absence of global genomic repair using rad7Δ mutants. We also determined whether the Rad26-independent transcription-coupled repair observed in rad26Δ and rad7Δ rad26Δ mutants depends on MSH2 by examining repair deficiencies of rad26Δ msh2Δ and rad7Δ rad26Δ msh2Δ mutants. We found no defects in transcription-coupled repair caused by mutations in the mismatch repair genes MSH2, MLH1, PMS1, and MSH3. Yeast appears to differ from bacteria and human cells in the capacity for transcription-coupled repair in a mismatch repair mutant background.
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Kraithong, Thanyalak, Silas Hartley, David Jeruzalmi, and Danaya Pakotiprapha. "A Peek Inside the Machines of Bacterial Nucleotide Excision Repair." International Journal of Molecular Sciences 22, no. 2 (January 19, 2021): 952. http://dx.doi.org/10.3390/ijms22020952.
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Double stranded DNA (dsDNA), the repository of genetic information in bacteria, archaea and eukaryotes, exhibits a surprising instability in the intracellular environment; this fragility is exacerbated by exogenous agents, such as ultraviolet radiation. To protect themselves against the severe consequences of DNA damage, cells have evolved at least six distinct DNA repair pathways. Here, we review recent key findings of studies aimed at understanding one of these pathways: bacterial nucleotide excision repair (NER). This pathway operates in two modes: a global genome repair (GGR) pathway and a pathway that closely interfaces with transcription by RNA polymerase called transcription-coupled repair (TCR). Below, we discuss the architecture of key proteins in bacterial NER and recent biochemical, structural and single-molecule studies that shed light on the lesion recognition steps of both the GGR and the TCR sub-pathways. Although a great deal has been learned about both of these sub-pathways, several important questions, including damage discrimination, roles of ATP and the orchestration of protein binding and conformation switching, remain to be addressed.
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Verhage, R. A., A. J. van Gool, N. de Groot, J. H. Hoeijmakers, P. van de Putte, and J. Brouwer. "Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair." Molecular and Cellular Biology 16, no. 2 (February 1996): 496–502. http://dx.doi.org/10.1128/mcb.16.2.496.
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The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.
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de Waard, Harm, Jan de Wit, Jaan-Olle Andressoo, Conny T. M. van Oostrom, Bente Riis, Allan Weimann, Henrik E. Poulsen, Harry van Steeg, Jan H. J. Hoeijmakers, and Gijsbertus T. J. van der Horst. "Different Effects of CSA and CSB Deficiency on Sensitivity to Oxidative DNA Damage." Molecular and Cellular Biology 24, no. 18 (September 15, 2004): 7941–48. http://dx.doi.org/10.1128/mcb.24.18.7941-7948.2004.
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ABSTRACT Mutations in the CSA and CSB genes cause Cockayne syndrome, a rare inherited disorder characterized by UV sensitivity, severe neurological abnormalities, and progeriod symptoms. Both gene products function in the transcription-coupled repair (TCR) subpathway of nucleotide excision repair (NER), providing the cell with a mechanism to remove transcription-blocking lesions from the transcribed strands of actively transcribed genes. Besides a function in TCR of NER lesions, a role of CSB in (transcription-coupled) repair of oxidative DNA damage has been suggested. In this study we used mouse models to compare the effect of a CSA or a CSB defect on oxidative DNA damage sensitivity at the levels of the cell and the intact organism. In contrast to CSB −/− mouse embryonic fibroblasts (MEFs), CSA −/− MEFs are not hypersensitive to gamma-ray or paraquat treatment. Similar results were obtained for keratinocytes. In contrast, both CSB −/− and CSA −/− embryonic stem cells show slight gamma-ray sensitivity. Finally, CSB −/− but not CSA −/− mice fed with food containing di(2-ethylhexyl)phthalate (causing elevated levels of oxidative DNA damage in the liver) show weight reduction. These findings not only uncover a clear difference in oxidative DNA damage sensitivity between CSA- and CSB-deficient cell lines and mice but also show that sensitivity to oxidative DNA damage is not a uniform characteristic of Cockayne syndrome. This difference in the DNA damage response between CSA- and CSB-deficient cells is unexpected, since until now no consistent differences between CSA and CSB patients have been reported. We suggest that the CSA and CSB proteins in part perform separate roles in different DNA damage response pathways.
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Citterio, Elisabetta, Vincent Van Den Boom, Gavin Schnitzler, Roland Kanaar, Edgar Bonte, Robert E. Kingston, Jan H. J. Hoeijmakers, and Wim Vermeulen. "ATP-Dependent Chromatin Remodeling by the Cockayne Syndrome B DNA Repair-Transcription-Coupling Factor." Molecular and Cellular Biology 20, no. 20 (October 15, 2000): 7643–53. http://dx.doi.org/10.1128/mcb.20.20.7643-7653.2000.
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ABSTRACT The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.
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Brosh, Robert M., Adayabalam S. Balajee, Rebecca R. Selzer, Morten Sunesen, Luca Proietti De Santis, and Vilhelm A. Bohr. "The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair." Molecular Biology of the Cell 10, no. 11 (November 1999): 3583–94. http://dx.doi.org/10.1091/mbc.10.11.3583.
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Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA andCSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, theCSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.
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Saijo, Masafumi, Tamami Hirai, Akiko Ogawa, Aki Kobayashi, Shinya Kamiuchi, and Kiyoji Tanaka. "Functional TFIIH Is Required for UV-Induced Translocation of CSA to the Nuclear Matrix." Molecular and Cellular Biology 27, no. 7 (January 22, 2007): 2538–47. http://dx.doi.org/10.1128/mcb.01288-06.
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ABSTRACT Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. Mutations in Cockayne syndrome group A and B genes (CSA and CSB) result in defective TCR, but the molecular mechanism of TCR in mammalian cells is not clear. We have found that CSA protein is translocated to the nuclear matrix after UV irradiation and colocalized with the hyperphosphorylated form of RNA polymerase II and that the translocation is dependent on CSB. We developed a cell-free system for the UV-induced translocation of CSA. A cytoskeleton (CSK) buffer-soluble fraction containing CSA and a CSK buffer-insoluble fraction prepared from UV-irradiated CS-A cells were mixed. After incubation, the insoluble fraction was treated with DNase I. CSA protein was detected in the DNase I-insoluble fraction, indicating that it was translocated to the nuclear matrix. In this cell-free system, the translocation was dependent on UV irradiation, CSB function, and TCR-competent CSA. Moreover, the translocation was dependent on functional TFIIH, as well as chromatin structure and transcription elongation. These results suggest that alterations of chromatin at the RNA polymerase II stall site, which depend on CSB and TFIIH at least, are necessary for the UV-induced translocation of CSA to the nuclear matrix.
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Sollier, Julie, Caroline Townsend Stork, María L. García-Rubio, Renee D. Paulsen, Andrés Aguilera, and Karlene A. Cimprich. "Transcription-Coupled Nucleotide Excision Repair Factors Promote R-Loop-Induced Genome Instability." Molecular Cell 56, no. 6 (December 2014): 777–85. http://dx.doi.org/10.1016/j.molcel.2014.10.020.
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van Oosterwijk, M. F., A. Versteeg, R. Filon, A. A. van Zeeland, and L. H. Mullenders. "The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes." Molecular and Cellular Biology 16, no. 8 (August 1996): 4436–44. http://dx.doi.org/10.1128/mcb.16.8.4436.
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Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells with either UV or NA-AAF, the basal transcription factor TFIIH becomes involved in nucleotide excision repair and that the CS gene products are involved in the conversion of TFIIH back to the transcription function. In this view, the CS proteins act as repair-transcription uncoupling factors. If the uncoupling process is defective, RNA synthesis will stay repressed, causing cellular sensitivity. Since transcription is essential for transcription-coupled repair, the CS defect will affect those lesions whose repair is predominantly transcription coupled, i.e., UV-induced cyclobutane pyrimidine dimers.
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Rodrigo, Gregory, Sophie Roumagnac, Marc S. Wold, Bernard Salles, and Patrick Calsou. "DNA Replication but Not Nucleotide Excision Repair Is Required for UVC-Induced Replication Protein A Phosphorylation in Mammalian Cells." Molecular and Cellular Biology 20, no. 8 (April 15, 2000): 2696–705. http://dx.doi.org/10.1128/mcb.20.8.2696-2705.2000.
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ABSTRACT Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.
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Reid-Bayliss, Kate S., Sarah T. Arron, Lawrence A. Loeb, Vladimir Bezrookove, and James E. Cleaver. "Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency." Proceedings of the National Academy of Sciences 113, no. 36 (August 19, 2016): 10151–56. http://dx.doi.org/10.1073/pnas.1610020113.
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Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.
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Gaillard, Hélène, Cristina Tous, Javier Botet, Cristina González-Aguilera, Maria José Quintero, Laia Viladevall, María L. García-Rubio, et al. "Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair." PLoS Genetics 5, no. 2 (February 6, 2009): e1000364. http://dx.doi.org/10.1371/journal.pgen.1000364.
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Luo, Chong, Hatice Osmanbeyoglu, Christina Leslie, and Ming Li. "Essential role of the Ets transcription factor GABP in control of T cell responses to antigen stimulation (IRM15P.454)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 199.2. http://dx.doi.org/10.4049/jimmunol.194.supp.199.2.
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Abstract Upon antigen stimulation, naive T cells transition from cellular quiescence into cell cycle progression and initiate functional differentiation. Such a coordinated response is orchestrated by numerous transcriptional programs, among which include Ets protein-dependent regulation. The Ets family of transcription factors, characterized by an evolutionarily conserved DNA-binding domain, is modulated by T cell receptor (TCR) signaling; yet the specific Ets member that is crucial for antigen receptor-mediated response remains elusive. GA-binding protein (GABP), which is composed of GABPα and GABPβ, is the only obligate multimeric complex among Ets factors. T cell-specific ablation of Gabpa gene in mice leads to a profound reduction in peripheral T cells. In response to antigen stimulation in vitro, GABPα-deficient T cells show diminished proliferation, dysregulation of reactive oxygen species and impaired cell survival. In addition, mice lacking GABPα fail to mount an antigen-specific T cell response to Listeria Monocytogenes infection. Transcriptome analysis coupled with chromatin immunoprecipitation sequencing identifies GABPα as a key regulator of the folate-dependent one carbon metabolism as well as cellular balance of redox. Our findings reveal critical functions of GABP-dependent transcriptional program in the control of TCR-stimulated metabolic reprogramming.
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Gomez-Rodriguez, Julio, Nisebita Sahu, Robin Handon, Maria Sacta, Stacie Anderson, Martha Kirby, Avery August, and Pamela Schwartzberg. "Differential expression of IL-17A and IL-17F is coupled to TCR signaling via Itk-mediated regulation of NFATc1 (139.4)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 139.4. http://dx.doi.org/10.4049/jimmunol.184.supp.139.4.
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Abstract Th17 CD4+ effector T cells play important roles in autoimmunity and responses to bacterial infections. The roles of cytokines in regulating the transcription factors STAT3 and RORγT in Th17 differentiation have been extensively studied. However, the role of T cell receptor (TCR) signaling in Th17 differentiation is less appreciated. To examine this issue, we evaluated IL17 production from T cells lacking Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ and downstream pathways. We find that Itk-/- CD4+T cells exhibit specific defects in the expression of IL-17A despite relatively normal expression of RORγT, RORα and the other Th17 cytokines, including the closely related IL-17F. Defects in IL-17A expression and differential regulation of IL-17A and IL-17F were also observed in vivo in Itk-/- mice challenged with an allergic asthma model. Although Itk-deficient cells have slightly depressed phosphorylation of STAT-3 in response to IL-6, expression of constitutively activated STAT-3 failed to rescue their IL-17A production. In contrast, expression of IL-17A could be rescued by pharmacologically-induced Ca2+ influx or by a constitutively active NFATc1. The effects of Itk on transcriptional complexes and chromatin at the IL-17 locus are under further investigation. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1 and that TCR signaling pathways differentially influence Th17 cytokine production.
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Smith, Martin L., James M. Ford, M. Christine Hollander, Rachel A. Bortnick, Sally A. Amundson, Young R. Seo, Chu-Xia Deng, Philip C. Hanawalt, and Albert J. Fornace. "p53-Mediated DNA Repair Responses to UV Radiation: Studies of Mouse Cells Lacking p53, p21, and/orgadd45 Genes." Molecular and Cellular Biology 20, no. 10 (May 15, 2000): 3705–14. http://dx.doi.org/10.1128/mcb.20.10.3705-3714.2000.
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ABSTRACT Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse embryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21(Cdkn1a), as well as MEFs lacking both gadd45and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss ofp53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wild-type MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.
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Shin, Samuel B., Bernard C. Lo, Maryam Ghaedi, R. Wilder Scott, Yicong Li, Melina Messing, Diana Canals Hernaez, et al. "Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors." Blood Advances 4, no. 21 (November 2, 2020): 5362–72. http://dx.doi.org/10.1182/bloodadvances.2020002758.
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Abstract Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.
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Duan, Mingrui, Kathiresan Selvam, John J. Wyrick, and Peng Mao. "Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin." Proceedings of the National Academy of Sciences 117, no. 31 (July 20, 2020): 18608–16. http://dx.doi.org/10.1073/pnas.2003868117.
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Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion ofSPT4significantly restores TC-NER across the genome in arad26∆mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.
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Menoni, Hervé, Jan H. J. Hoeijmakers, and Wim Vermeulen. "Nucleotide excision repair–initiating proteins bind to oxidative DNA lesions in vivo." Journal of Cell Biology 199, no. 7 (December 17, 2012): 1037–46. http://dx.doi.org/10.1083/jcb.201205149.
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Base excision repair (BER) is the main repair pathway to eliminate abundant oxidative DNA lesions such as 8-oxo-7,8-dihydroguanine. Recent data suggest that the key transcription-coupled nucleotide excision repair factor (TC-NER) Cockayne syndrome group B (CSB) and the global genome NER-initiating factor XPC are implicated in the protection of cells against oxidative DNA damages. Our novel live-cell imaging approach revealed a strong and very rapid recruitment of XPC and CSB to sites of oxidative DNA lesions in living cells. The absence of detectable accumulation of downstream NER factors at the site of local oxidative DNA damage provide the first in vivo indication of the involvement of CSB and XPC in the repair of oxidative DNA lesions independent of the remainder of the NER reaction. Interestingly, CSB exhibited different and transcription-dependent kinetics in the two compartments studied (nucleolus and nucleoplasm), suggesting a direct transcription-dependent involvement of CSB in the repair of oxidative lesions associated with different RNA polymerases but not involving other NER proteins.
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Wu, J., S. Katzav, and A. Weiss. "A functional T-cell receptor signaling pathway is required for p95vav activity." Molecular and Cellular Biology 15, no. 8 (August 1995): 4337–46. http://dx.doi.org/10.1128/mcb.15.8.4337.
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Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade.
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Zhang, Yaguang, Qin Zhang, Yang Zhang, and Junhong Han. "The Role of Histone Modification in DNA Replication-Coupled Nucleosome Assembly and Cancer." International Journal of Molecular Sciences 24, no. 5 (March 3, 2023): 4939. http://dx.doi.org/10.3390/ijms24054939.
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Histone modification regulates replication-coupled nucleosome assembly, DNA damage repair, and gene transcription. Changes or mutations in factors involved in nucleosome assembly are closely related to the development and pathogenesis of cancer and other human diseases and are essential for maintaining genomic stability and epigenetic information transmission. In this review, we discuss the role of different types of histone posttranslational modifications in DNA replication-coupled nucleosome assembly and disease. In recent years, histone modification has been found to affect the deposition of newly synthesized histones and the repair of DNA damage, further affecting the assembly process of DNA replication-coupled nucleosomes. We summarize the role of histone modification in the nucleosome assembly process. At the same time, we review the mechanism of histone modification in cancer development and briefly describe the application of histone modification small molecule inhibitors in cancer therapy.
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Hu, Jinchuan, Jun-Hyuk Choi, Shobhan Gaddameedhi, Michael G. Kemp, Joyce T. Reardon, and Aziz Sancar. "Nucleotide Excision Repair in Human Cells." Journal of Biological Chemistry 288, no. 29 (June 8, 2013): 20918–26. http://dx.doi.org/10.1074/jbc.m113.482257.
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Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.
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Agapov, Aleksei, Anna Olina, and Andrey Kulbachinskiy. "RNA polymerase pausing, stalling and bypass during transcription of damaged DNA: from molecular basis to functional consequences." Nucleic Acids Research 50, no. 6 (March 22, 2022): 3018–41. http://dx.doi.org/10.1093/nar/gkac174.
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Abstract Cellular DNA is continuously transcribed into RNA by multisubunit RNA polymerases (RNAPs). The continuity of transcription can be disrupted by DNA lesions that arise from the activities of cellular enzymes, reactions with endogenous and exogenous chemicals or irradiation. Here, we review available data on translesion RNA synthesis by multisubunit RNAPs from various domains of life, define common principles and variations in DNA damage sensing by RNAP, and consider existing controversies in the field of translesion transcription. Depending on the type of DNA lesion, it may be correctly bypassed by RNAP, or lead to transcriptional mutagenesis, or result in transcription stalling. Various lesions can affect the loading of the templating base into the active site of RNAP, or interfere with nucleotide binding and incorporation into RNA, or impair RNAP translocation. Stalled RNAP acts as a sensor of DNA damage during transcription-coupled repair. The outcome of DNA lesion recognition by RNAP depends on the interplay between multiple transcription and repair factors, which can stimulate RNAP bypass or increase RNAP stalling, and plays the central role in maintaining the DNA integrity. Unveiling the mechanisms of translesion transcription in various systems is thus instrumental for understanding molecular pathways underlying gene regulation and genome stability.
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Martinez, Ernest, Vikas B. Palhan, Agneta Tjernberg, Elena S. Lymar, Armin M. Gamper, Tapas K. Kundu, Brian T. Chait, and Robert G. Roeder. "Human STAGA Complex Is a Chromatin-Acetylating Transcription Coactivator That Interacts with Pre-mRNA Splicing and DNA Damage-Binding Factors In Vivo." Molecular and Cellular Biology 21, no. 20 (October 15, 2001): 6782–95. http://dx.doi.org/10.1128/mcb.21.20.6782-6795.2001.
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ABSTRACT GCN5 is a histone acetyltransferase (HAT) originally identified inSaccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAFII-containing complex], and STAGA [SPT3-TAFII31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.
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Xu, Jun, Wei Wang, Liang Xu, Jia-Yu Chen, Jenny Chong, Juntaek Oh, Andres E. Leschziner, Xiang-Dong Fu, and Dong Wang. "Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers." Proceedings of the National Academy of Sciences 117, no. 41 (September 28, 2020): 25486–93. http://dx.doi.org/10.1073/pnas.2013379117.
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While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.
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Packard, Jessica E., and Jill A. Dembowski. "HSV-1 DNA Replication—Coordinated Regulation by Viral and Cellular Factors." Viruses 13, no. 10 (October 7, 2021): 2015. http://dx.doi.org/10.3390/v13102015.
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DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and recombination of the viral genome, and viral gene transcription. HSV-1 encodes its own DNA replication machinery, including an origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), and a helicase/primase complex (UL5/UL8/UL52). In addition, HSV-1 utilizes a combination of accessory viral and cellular factors to coordinate viral DNA replication with other viral and cellular processes. The purpose of this review is to outline the roles of viral and cellular proteins in HSV-1 DNA replication and replication-coupled processes, and to highlight how HSV-1 may modify and adapt cellular proteins to facilitate productive infection.
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Xu, Jun, Jenny Chong, and Dong Wang. "Strand-specific effect of Rad26 and TFIIS in rescuing transcriptional arrest by CAG trinucleotide repeat slip-outs." Nucleic Acids Research 49, no. 13 (July 1, 2021): 7618–27. http://dx.doi.org/10.1093/nar/gkab573.
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Abstract Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.
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Morel, Penelope A., William F. Hawse, Robert P. Sheehan, William C. Boggess, and James R. Faeder. "TCR signal strength regulates Akt substrate specificity to induce alternate Th and Treg differentiation programs." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 128.10. http://dx.doi.org/10.4049/jimmunol.196.supp.128.10.
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Abstract T regulatory (Treg) cells are induced following stimulation of naïve CD4 T cells with low dose antigen to an extent that is negatively correlated with signaling via the Akt/mTOR pathway. Strong TCR signals induce high levels of Akt activity that inhibit development of Treg by poorly understood mechanisms. Here, we show that high dose stimulation of T cells results in the phosphorylation of Akt on two regulatory sites, Serine (S) 473 and Threonine (T) 308, whereas low dose stimulation results in only T308 phosphorylation. Mathematical modeling shows that the phosphorylation of Akt on both S473 and T308 is controlled by a feedback loop involving PTEN, mTORC2 and the transcription factor FoxO1 that creates a sharp activation threshold with respect to antigen dose and stimulus duration. Using mass spectrometry to analyze phosphorylated Akt substrates at different levels of stimulation, we find profound differences in the substrates phosphorylated, suggesting that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates. Proteins differentially phosphorylated by these two states of Akt include RNA splicing factors, and we find changes in the splice variant expression levels of key TCR signaling proteins, such as CD3ζ and CD45 that correlate with the observed differences in cell fate. Knockdown of specific splicing factors impacted the ratios of Th versus Treg cells induced. Together, this work demonstrates that alternative splicing can affect the outcome of T cell fate decisions and identifies alternate Akt-mediated signaling networks that drive CD4+ T cell differentiation.
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Yedidia-Aryeh, Lia, and Michal Goldberg. "The Interplay between the Cellular Response to DNA Double-Strand Breaks and Estrogen." Cells 11, no. 19 (October 1, 2022): 3097. http://dx.doi.org/10.3390/cells11193097.
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Cancer development is often connected to impaired DNA repair and DNA damage signaling pathways. The presence of DNA damage in cells activates DNA damage response, which is a complex cellular signaling network that includes DNA repair, activation of the cell cycle checkpoints, cellular senescence, and apoptosis. DNA double-strand breaks (DSBs) are toxic lesions that are mainly repaired by the non-homologous end joining and homologous recombination repair (HRR) pathways. Estrogen-dependent cancers, like breast and ovarian cancers, are frequently associated with mutations in genes that play a role in HRR. The female sex hormone estrogen binds and activates the estrogen receptors (ERs), ERα, ERβ and G-protein-coupled ER 1 (GPER1). ERα drives proliferation, while ERβ inhibits cell growth. Estrogen regulates the transcription, stability and activity of numerus DDR factors and DDR factors in turn modulate ERα expression, stability and transcriptional activity. Additionally, estrogen stimulates DSB formation in cells as part of its metabolism and proliferative effect. In this review, we will present an overview on the crosstalk between estrogen and the cellular response to DSBs. We will discuss how estrogen regulates DSB signaling and repair, and how DDR factors modulate the expression, stability and activity of estrogen. We will also discuss how the regulation of HRR genes by estrogen promotes the development of estrogen-dependent cancers.
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Liu, Haoxuan, and Jianzhi Zhang. "Higher Germline Mutagenesis of Genes with Stronger Testis Expressions Refutes the Transcriptional Scanning Hypothesis." Molecular Biology and Evolution 37, no. 11 (July 8, 2020): 3225–31. http://dx.doi.org/10.1093/molbev/msaa168.
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Abstract Why are more genes expressed in the testis than in any other organ in mammals? The recently proposed transcriptional scanning hypothesis posits that transcription alleviates mutagenesis through transcription-coupled repair so has been selected in the testis to modulate the germline mutation rate in a gene-specific manner. Here, we show that this hypothesis is theoretically untenable because the selection would be too weak to have an effect in mammals. Furthermore, the analysis purported to support the hypothesis did not control known confounding factors and inappropriately excluded genes with no observed de novo mutations. After remedying these problems, we find the human germline mutation rate of a gene to rise with its testis expression level. This trend also exists for inferred coding strand-originated mutations, suggesting that it arises from transcription-associated mutagenesis. Furthermore, the testis expression level of a gene robustly correlates with its overall expression in other organs, nullifying the need to explain the testis silencing of a minority of genes by adaptive germline mutagenesis. Taken together, our results demonstrate that human testis transcription increases the germline mutation rate, rejecting the transcriptional scanning hypothesis of extensive gene expressions in the mammalian testis.
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Moriel-Carretero, María, Sara Ovejero, Marie Gérus-Durand, Dimos Vryzas, and Angelos Constantinou. "Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors." Journal of Cell Biology 216, no. 12 (October 13, 2017): 4007–26. http://dx.doi.org/10.1083/jcb.201702136.
Full textAbstract:
Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes.
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Zisa, David, Arsalan Shabbir, Michalis Mastri, Tyler Taylor, Ilija Aleksic, Mary McDaniel, Gen Suzuki, and Techung Lee. "Intramuscular VEGF activates an SDF1-dependent progenitor cell cascade and an SDF1-independent muscle paracrine cascade for cardiac repair." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 6 (December 2011): H2422—H2432. http://dx.doi.org/10.1152/ajpheart.00343.2011.
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The skeletal muscle is endowed with an impressive ability to regenerate after injury, and this ability is coupled to paracrine production of many trophic factors possessing cardiovascular benefits. Taking advantage of this humoral capacity of the muscle, we recently demonstrated an extracardiac therapeutic regimen based on intramuscular delivery of VEGF-A165 for repair of the failing hamster heart. This distal organ repair mechanism activates production from the injected hamstring of many trophic factors, among which stromal-derived factor-1 (SDF1) prominently mobilized multi-lineage progenitor cells expressing CXCR4 and their recruitment to the heart. The mobilized bone marrow progenitor cells express the cardiac transcription factors myocyte enhancer factor 2c and GATA4 and several major trophic factors, most notably IGF1 and VEGF. SDF1 blockade abrogated myocardial recruitment of CXCR4+ and c-kit+ progenitor cells with an insignificant effect on the hematopoietic progenitor lineage. The knockdown of cardiac progenitor cells led to deprivation of myocardial trophic factors, resulting in compromised cardiomyogenesis and angiogenesis. However, the VEGF-injected hamstring continued to synthesize cardioprotective factors, contributing to moderate myocardial tissue viability and function even in the presence of SDF1 blockade. These findings thus uncover two distinct but synergistic cardiac therapeutic mechanisms activated by intramuscular VEGF. Whereas the SDF1/CXCR4 axis activates the progenitor cell cascade and its trophic support of cardiomyogenesis intramuscularly, VEGF amplifies the skeletal muscle paracrine cascade capable of directly promoting myocardial survival independent of SDF1. Given that recent clinical trials of cardiac repair based on the use of marrow-mobilizing agents have been disappointing, the proposed dual therapeutic modality warrants further investigation.
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Onabote, Oladapo, Haider M. Hassan, Majdina Isovic, and Joseph Torchia. "The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer." Cancers 14, no. 3 (February 1, 2022): 765. http://dx.doi.org/10.3390/cancers14030765.
Full textAbstract:
DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of Tdg and the onset of tumorigenesis. In this review, we summarize the recent findings on TDG’s function as a transcriptional regulator as well as the physiological relevance of TDG and active DNA demethylation in cancer.
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Yang, Lili, Adam W. Mailloux, Dana E. Rollison, Jong Park, Jeffrey S. Painter, Rami S. Komrokji, Jaroslaw P. Maciejewski, et al. "Human Telomerase Reverse Transcriptase (hTERT) Deficiency in Myelodysplastic Syndrome (MDS) Demonstrates Mechanistic Linkage to Aplastic Anemia Pathophysiology." Blood 118, no. 21 (November 18, 2011): 791. http://dx.doi.org/10.1182/blood.v118.21.791.791.
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Abstract Abstract 791 Background: Myelodysplastic syndromes (MDS) are characterized by dysregulated myelopoiesis and peripheral cytopenias with enormous disease heterogeneity owing to diverse molecular pathobiology. The early manifestations of MDS, however, are relatively well conserved and include increased apoptosis coupled to excessive proliferation of myeloid progenitors. In addition to myeloid abnormalities, repertoire contraction and memory expansion is demonstrable in T cells. The notion that apoptosis of hematopoetic cells may be triggered through an immune–mediated mechanism arose from similarities with aplastic anemia (AA). Our recent data showed that MDS responsive to immunosuppressive therapy has accelerated naïve T cell turnover (ie, high proliferative index plus excessive cell death) which led us to hypothesize the presence of an inherent T cell abnormality impairing homeostatic regulation. AA can be caused by somatic mutations within telomere repair components. T-cells are one of a few somatic cells that retain telomerase function to control naïve T-cell survival, replication potential, and antigenic diversity. To this end, we examined telomere function and replicative burst capacity of MDS T cells as a possible mechanism for immune dysregulation. Methods: Primary specimens from MDS (n=37), AA (n=8), and controls (n=42) were investigated. Peripheral blood mononuclear cells were isolated from patient blood or buffy coats by Ficoll-Hypaque gradient centrifugation. Purified CD3+ T cells were isolated using negative selection and then stimulated with anti-CD3/anti-CD28 T cell activator beads (Dynabead®) for 3 days. Telomere length was assessed by quantitative PCR (q-PCR) and telomerase enzymatic function measured by Telomere Repeat Amplification Protocol (TRAP) assays. Results: Mean telomere length in purified T cells was significantly shorter among MDS patients compared to controls after adjusting for age and sex (p<0.0001). To assess telomerase repair function in MDS T-cells, we performed TRAP assays with purified T cells after stimulation and found that inducible telomerase activity is severely suppressed in MDS compare to controls. In comparison to controls, the inducible telomerase activity fell below the 95% confidence internal in all cases (MDS median 18.70, 95% CI, 15.93–20.54 vs control median 45.0, 95% CI, 45.79 – 64.5, p<0.0001) and the amount of telomerase activity was unrelated to risk stratification by the International Prognostic Scoring System (IPSS), World Health Organization (WHO) classification, and age indicating that it is a frequent abnormality in the disease. Analysis of telomerase function and telomere length in T cells from patients with AA showed a similar deficiency in telomerase repair function. The mechanism responsible for telomerase insufficiency in MDS was mediated by defective induction of telomerase reverse transcriptase (hTERT) transcription; the key enzyme involved in telomere maintenance. Next, to determine the functional consequences of the disturbance in telomere repair in MDS, the ability of T cells to enter S-phase and to undergo an antigen-induced proliferative burst were examined. TCR signaling was shown to be preserved, evidenced by induction of an early activation antigen CD69. Although some cells were capable of entering S-phase, the replicative burst potential was severely impaired in T cells form all patients. Telomere repair is exclusively present in naïve T cells and progressively declines after memory transition. TCR triggered telomerase activity was measured in sorted naïve (CD45RA+, CD45RO-) and memory (CD45RO+, CD45RA-) T cells. The telomere length in naïve cells was shorter in MDS patients compared to controls (p=0.018) and the telomerase activity was suppressed in naïve MDS T cells (p=0.0207) indicating that telomere dysfunction underlies the altered homeostasis of naïve T cells in MDS, a feature mechanistically akin to AA and other telomere repair disorders. Conclusion: Results of this study indicate that there is loss of telomere maintenance in naïve T cells due to a defect in hTERT transcription is associated with impaired replicative potential. This abnormality in naïve T cell homeostasis represents an inherent defect that contributes to a memory cell growth advantage and repertoire contraction associated with autoimmunity in AA and MDS. Disclosures: No relevant conflicts of interest to declare.
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Dammann, R., and G. P. Pfeifer. "Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes." Molecular and Cellular Biology 17, no. 1 (January 1997): 219–29. http://dx.doi.org/10.1128/mcb.17.1.219.
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UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.
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Debnath, Sandip, Achal Kant, Pradipta Bhowmick, Ayushman Malakar, Shampa Purkaystha, Binod Kumar Jena, Gaurav Mudgal, et al. "The Enhanced Affinity of WRKY Reinforces Drought Tolerance in Solanum lycopersicum L.: An Innovative Bioinformatics Study." Plants 12, no. 4 (February 8, 2023): 762. http://dx.doi.org/10.3390/plants12040762.
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In the scenario of global climate change, understanding how plants respond to drought is critical for developing future crops that face restricted water resources. This present study focuses on the role of WRKY transcription factors on drought tolerance in tomato, Solanum lycopersicum L., which is a significant vegetable crop. WRKY transcription factors are a group of proteins that regulate a wild range of growth and developmental processes in plants such as seed germination and dormancy and the stress response. These transcription factors are defined by the presence of a DNA-binding domain, namely, the WRKY domain. It is well-known that WRKY transcription factors can interact with a variety of proteins and therefore control downstream activities. It aims to simulate the effect of curcumin, a bioactive compound with regulatory capacity, on the protein–protein interaction events by WRKY transcription factors with an emphasis on drought stress. It was found that curcumin binds to WRKY with an energy of −11.43 kcal/mol with inhibitory concentration (Ki) 0.12 mM and has the potential to improve fruit quality and reinforce drought tolerance of S. lycopersicum, according to the results based on bioinformatics tools. The root means square deviation (RMSD) of the C-α, the backbone of 2AYD with ligand coupled complex, displayed a very stable structure with just a little variation of 1.89 Å. MD simulation trajectory of Cα atoms of 2AYD bound to Curcumin revealed more un-ordered orientation in PC1 and PC10 modes and more toward negative correlation from the initial 400 frames during PCA. Establishing the binding energies of the ligand–target interaction is essential in order to characterize the compound’s binding affinity to the drought transcription factor. We think we have identified a phyto-agent called curcumin that has the potential to enhance the drought tolerance. Compared to the part of the mismatch repair-base technique that can be used to fix drought related genes, curcumin performed better in a drop-in crop yield over time, and it was suggested that curcumin is a potential candidate factor for improving drought tolerance in tomatoes, and it needs future validation by experiments in laboratory and field.
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Rubtsov, Yury, Кirill Goryunov, Аndrey Romanov, Yulia Suzdaltseva, George Sharonov, and Vsevolod Tkachuk. "Molecular Mechanisms of Immunomodulation Properties of Mesenchymal Stromal Cells: A New Insight into the Role of ICAM-1." Stem Cells International 2017 (2017): 1–15. http://dx.doi.org/10.1155/2017/6516854.
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Mesenchymal stromal cells (MSC) control excessive inflammation and create a microenvironment for tissue repair protecting from chronic inflammation and tissue fibrosis. We examined the molecular mechanisms of MSC immunomodulatory function in mixed cultures of human adipose-derived MSC with lymphocytes. Our data show that MSC promote unstimulated lymphocyte survival potentially by an increase in antigen presentation. Under inflammatory conditions, mimicked by stimulation of TCR in lymphocytes, MSC suppress activation and proliferation of stimulated T cells. Immunosuppression is accompanied by downregulation of IL-2Rαthat negatively affects the survival of activated T cells. MSC upregulate transcription of indolamine-2,3-dioxygenase (IDO) and inducible NO synthase (iNOS), which generate products negatively affecting T cell function. Both MSC and lymphocytes dramatically increase the surface ICAM-1 level in mixed cultures. Antibody-mediated blockage of surface ICAM-1 partially releases MSC-mediated immune suppression in vitro. Our data suggest that MSC have cell-intrinsic molecular programs depending on the inflammatory microenvironment. We speculate that MSC sense soluble factors and respond by surface ICAM-1 upregulation. ICAM-1 is involved in the control of T cell activation leading to immunosuppression or modest stimulation depending on the T cell status. Immunomodulation by MSC ranging from support of naive T cell survival to immunosuppression of activated T cells may affect the tissue microenvironment protecting from aberrant regeneration.
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Akhter, Md Zahid, Jagdish Chandra Joshi, Vijay Avin Balaji Ragunathrao, Mark Maienschein-Cline, Richard L. Proia, Asrar B. Malik, and Dolly Mehta. "Programming to S1PR1 + Endothelial Cells Promotes Restoration of Vascular Integrity." Circulation Research 129, no. 2 (July 9, 2021): 221–36. http://dx.doi.org/10.1161/circresaha.120.318412.
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Rationale: Increased endothelial permeability and defective repair are the hallmarks of several vascular diseases, including acute lung injury. However, little is known about the intrinsic pathways activating the endothelial cell (EC) regenerative programs. Objective: Studies have invoked a crucial role of S1P (sphingosine-1-phosphate) in resolving endothelial hyperpermeability through the activation of the GPCR (G-protein–coupled receptor), S1PR1 (S1P receptor 1). Here, we addressed mechanisms of generation of a population of S1PR1 + EC and their pivotal role in restoring endothelial integrity. Methods and Results: Studies were made using inducible EC-S1PR1 −/− ( iEC-S1PR1 −/− ) mice and S1PR1-GFP (green fluorescent protein) reporter mice to trace the generation of S1PR1 + EC. We observed in a mouse model of endotoxemia that S1P generation induced the programming of S1PR1 lo to S1PR1 + EC, which eventually comprised 80% of the lung EC. The cell transition was required for reestablishing the endothelial junctional barrier. We observed that conditional deletion of S1PR1 in EC increased endothelial permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, specifically SPHK1 (sphingosine kinase 1) and SPNS2 (sphingolipid transporter 2). Activation of transcription factors EGR1 (early growth response 1) and STAT3 (signal transducer and activator of transcription 3) was required for transcribing SPHK1 and SPNS2, respectively, and both served to increase S1P production and amplify S1PR1 + EC transition. Furthermore, transplantation of S1PR1 + EC population into injured lung vasculature restored endothelial integrity. Conclusions: Our findings show that generation of the S1PR1 + EC population activates the endothelial regenerative program to mediate endothelial repair. Results raise the possibility of harnessing this pathway to restore vascular homeostasis in inflammatory vascular injury states.