TFIIH mutants treated with methyl methanesulfonate
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE2343 | GEO | 2005/04/20
SECONDARY ACCESSION(S): PRJNA91569
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose of profiling experiment was to examine effects of a mutation of a subunit of TFIIH, ssl1p, which has ubiquitin ligase activity.,The whole complex has previously been shown to have both kinase and helicase activities. We wanted to see if 1) there were,general transcriptional defects with these mutations, as there are with other mutations to kinase activity, and 2) if there,were transcriptional effects on DNA repair machinery when the mutants were treated with MMS, which is an alkylating agent.
2007-07-08 | E-GEOD-2343 | biostudies-arrayexpress
Project description:Humans are exposed to a wide range of exogenous and endogenous alkylating agents, including agents frequently used as cytostatic drugs in cancer treatment (Drablos, Feyzi et al. 2004). Alkylating agents are mutagenic and cytotoxic and the major body of research has focused on their effects upon DNA. Less is known about the cellular consequences of alkylation to RNA, proteins and lipids, although such damage is likely quantitatively dominating due to the mere abundance of these macromolecules. DNA and RNA can be alkylated by SN1 and SN2 alkylating agents and alkylation occurs at nucleophilic O and N-atoms in the nucleosides as well as at O atoms in the phosphodiester bonds (Shooter, Howse et al. 1974, Sedgwick 2004). While alkylation at the O atoms in the nucleobases (O6G, O4T and O2C) are not affected by their hydrogen bonding, N1A and N3C contain an electron pair involved in hydrogen bonding. Alkylation at these nitrogens are thus more frequent in RNA and single-stranded DNA than in double stranded DNA (Bodell and Singer 1979). To cope with genomic damage inflicted by alkylating agents, a set of complex signal transduction pathways and DNA repair enzymes, collectively called the DNA damage response (DDR) is essential. The DDR senses the DNA damage and starts a highly choreographed response in order to resolve DNA damage or -replication issues (Ciccia and Elledge 2010). Specific repair mechanisms also exist to repair alkylated RNA (Aas, Otterlei et al. 2003, Wurtmann and Wolin 2009), although the biological significance of such repair yet remains elusive. Nevertheless, alkylation damage has been shown to alter the base-pairing properties of RNA bases and can interfere with tRNA-rRNA (Yoshizawa, Fourmy et al. 1999) as well as mRNA-tRNA (Ougland, Zhang et al. 2004){Hudson, 2015 #259}. Likely, other RNA functions that relies on base pairing might also be affected by alkylation damage, e.g. the correct folding of tRNAs and the function of miRNAs. Alkylation of mRNA may also affect translation indirectly by affecting binding affinities to RNA binding proteins (RBPs), as recently demonstrated for 6-meA. This enzymatically induced modification promotes binding of the 6-maA reader proteins YTHDF1 and 2 . While YTHDF2 reduces the stability of 6-maA modified transcripts (Wang, Lu et al. 2014), YTHDF1 actively promotes protein synthesis by interacting with the translation machinery (Wang, Zhao et al. 2015). To what degree alkylation damage to mRNA may promote similar effects by modulating translation remains, however, to be investigated. Interestingly, several studies have identified RBPs as important players in the DDR (Beli, Lukashchuk et al. 2012, Jungmichel, Rosenthal et al. 2013). Here, RBPs appear to have three major roles: 1) regulate gene expression at both the transcriptional- and post-transcriptional level (Keene and Tenenbaum 2002, Glisovic, Bachorik et al. 2008, Rinn and Chang 2012), 2) prevent formation of R-loops (Skourti-Stathaki and Proudfoot 2014), and 3) participate directly in DNA repair (Montecucco and Biamonti 2013, Dutertre, Lambert et al. 2014, Naro, Bielli et al. 2015). Two large-scale mass spectrometry (MS) based studies of RBPs in human cancer cell lines together identified more than 1100 RBPs (Baltz, Munschauer et al. 2012, Castello, Fischer et al. 2012) and more recently the “RBPomes” of mouse embryonic stem cells (Kwon, Yi et al. 2013) and yeast (Mitchell, Jain et al. 2013) have been published. However, functional studies describing how these complex protein networks are affected during genotoxic cell stress are still largely missing. One previous study has monitored changes in the RBPome of mouse embryonic fibroblasts subsequent to treatment with the topoisomerase inhibitor etoposide {Boucas, 2015 #112}. To our knowledge, however, no such studies have attempted to monitor altered global protein:RNA binding subsequent to treatment with alkylating agents. This could be particularly interesting since base methylation constitutes an important functional modification of RNA. In the present study we aimed at investigating this in more detail by SILAC-based quantitation of proteins differentially binding to poly(A)-RNA in HeLa cells at different time points after treatment with the alkylating agent methyl methanesulfonate.
2021-08-30 | PXD003549 | Pride
Project description:We explored the global transcription profile in response to MMS in C. albicans through RNAseq assay. Here, we identified 407 genes (log2 fold cut off 1.0, P<0.05) whose levels of transcription were significantly affected under the MMS treatment condition; 207 genes were upregulated, and 200 genes were down-regulated. GO term analysis revealed that a small portion of changed genes showed direct roles in DNA repair progress, but a large number of induced genes are involved in antioxidant responses, and some down-regulated genes are involved in nucleosome packing and nitrogen utilization. In addition, the changed transcription of some DNA repair genes, as well as antioxidants genes, are blocked by deleting RAD53. Taken together, treatment of the DNA damage agent MMS induces a global transcriptional change in C. albicans, but not only DNA damage response genes.
2022-02-10 | GSE196244 | GEO
Project description:To determine the effect of lack of two Histone H4 genes, CgHHF1 and CgHHF2, on global transcriptional profile, and on transcriptional response towards MMS exposure, RNA-Seq analysis was conducted on log-phase grown Candida glabrata wild-type and Cghhf1∆2∆ cells in the presence and absence of MMS. Comparative transcriptome analysis of wild-type and Cghhf1∆2∆ cells displayed deregulation of 303 genes due to the loss of CgHHF1 and CgHHF2 genes. C. glabrata responded to MMS by upregulating genes involved in transmembrane transport and invasive growth in response to glucose limitation, and by downregulating the aerobic respiration and ergosterol biosynthetic process genes.
2020-02-20 | GSE142737 | GEO
Project description:Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a ‘plug’ element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.
2019-07-02 | PXD013947 | Pride
Project description:CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e. distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a “master regulator” role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g. SPT5, SF3B1) are phosphorylated by the 3-subunit CDK-activating kinase (CAK; CCNH, MAT1, CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
2020-10-31 | GSE151699 | GEO
Project description:Understanding the mechanisms by which cells respond to chemotherapeutics is key to identifying means to improve therapy effiicacy while reducing systemic toxicity of these widely used classes of drugs. While determining the role of NRF2-GSH and ER stress in cells exposed to alkylating compounds such as methyl-methanesulfonate (MMS), we asked if these pathways could also be a general cell damage response relevant to other clinically used chemotherapeutics or if it is an alkylation specific response. With this intent, we performed RNA sequencing of MDA-MB231 breast cancer and U2OS osteosarcoma cells lines treated for 8 hours with a topoisomerase II inhibitor etoposide (20 µM), the antimitotic beta-tubulin-interacting drug paclitaxel (0.2 µM), doxorubicin (1 µM) and compared to MMS (40 µg/mL) treated cells. Doses represent IC50 level after 72 hours exposure. We observed that even though non-alkylating drugs, especially etoposide, caused an increase in the mRNA expression of some NRF2 and ER stress signaling markers, the number and magnitude of upregulation of genes markers in either pathway was more pronounced in alkylation treatments compared to other drugs. This indicates that alterations in NRF2 and ER stress pathways could be more likely associated with differential sensitivity to alkylating chemotherapies.
2017-02-13 | GSE84863 | GEO
Project description:The initial step of RNA polymerase II (Pol II) transcription involves a large number of transcription factors and arises at multiple sites within most promoters. TFIIH is an essential, multi-subunit transcription factor that assembles on promoter DNA with Pol II and five other general transcription factors (GTFs) to form a pre-initiation complex (PIC) for basal transcription. During transcription initiation, TFIIH melts promoter DNA through the ATPase activity of its Ssl2 subunit. In the model eukaryote Saccharomyces cerevisiae, after DNA melting, Pol II scans downstream for usable transcription start sites (TSSs). To understand the function of Ssl2/TFIIH in promoter scanning and TSS selection, we identified novel alleles of SSL2 in genetic screens for mutants defective in TSS distribution that may potentially arise from altered scanning. Consistent with this notion, these ssl2 alleles alter scanning in ways that are distinct from how changes to the Pol II active site alter scanning and this difference is observed genome-wide. Our investigations support two major pathways in controlling promoter scanning and TSS selection, one controlling the efficiency of initiation through Pol II activity or factors regulating Pol II activity; another network appears to control the processivity of scanning by Ssl2/TFIIH.
2021-08-28 | GSE182792 | GEO
Project description:To obtain a better understanding of molecular mechanisms underlying the DNA damage response in Δckb1 and Δckb2 mutants, transcriptome analyses were performed between WT, Δckb1, and Δckb2 strains. Comparative transcriptome analysis of Δckb1 and Δckb2 mutants showed similar transcriptional profiles in the presence and absence of MMS. The data also indicated that compared with WT, about 200 genes were up or downregulated in Δckb1 and Δckb2 strains with or without treatment with 0.01% MMS. GO analysis highlighted that the differentially expressed genes in Δckb1 and Δckb2 were significantly enriched in the processes related to the programmed formation of DNA double-strand breaks (DSBs), recombination, and DNA repair coordination of the meiotic cell cycle.
2023-10-11 | GSE232439 | GEO
Project description:The 10-subunit TFIIH complex is vital to transcription and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental disorder trichothiodystrophy. Some trichothiodystrophy features are thought to be caused by subtle transcription or gene expression defects. TTDA/GTF2H5 knockout mice are not viable, making it difficult to investigate TTDA/GTF2H5 in vivo function. Here we show that deficiency of C. elegans TTDA ortholog GTF-2H5 is, however, compatible with life, in contrast to depletion of other TFIIH subunits. GTF-2H5 promotes TFIIH stability in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of TFIIH to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as model for studying pathogenesis of this disease.
2021-11-19 | PXD029008 | Pride