C. elegans survival assays to discern global and transcription-coupled nucleotide excision repair.
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ABSTRACT:
SUBMITTER: van der Woude M
PROVIDER: S-EPMC8192855 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that is regulated by multiple facilitators, such as Rad26, and repressors, such as Rpb4 and Spt4/Spt5. How these factors interplay with each other and with core RNA polymerase II (RNAPII) remains largely unknown. In this study, we identified Rpb7, an essential RNAPII subunit, as another TCR repressor and characterized its repression of TCR in the AGP2, RPB2, and YEF3 genes, which are transcribed at low, moderate, and high rates, respectively. The Rpb7 region that interacts with the KOW3 domain of Spt5 represses TCR largely through the same common mechanism as Spt4/Spt5, as mutations in this region mildly enhance the derepression of TCR by spt4Δ only in the YEF3 gene but not in the AGP2 or RPB2 gene. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII repress TCR largely independently of Spt4/Spt5, as mutations in these regions synergistically enhance the derepression of TCR by spt4Δ in all the genes analyzed. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII may also play positive roles in other (non-NER) DNA damage repair and/or tolerance mechanisms, as mutations in these regions can cause UV sensitivity that cannot be attributed to derepression of TCR. Our study reveals a novel function of Rpb7 in TCR regulation and suggests that this RNAPII subunit may have broader roles in DNA damage response beyond its known function in transcription.
| S-EPMC10382679 | biostudies-literature
Project description:UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.
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Project description:Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3-H4 tetramer and two H2A-H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions.
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Project description:Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG-NER), which is responsible for repair throughout genomes, and transcription-coupled NER (TC-NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC-NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter-strand cross-link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.
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Project description:Repair of UV damage from the transcribed strand (TS) of yeast genes is rapid due to the transcription coupled nucleotide excision repair (TC-NER) pathway. TC-NER is triggered when RNA polymerase stalls at UV damage, such as a UV-induced cyclobutane pyrimidine dimer (CPD). During transcription, the histone methyltransferase Set2 methylates histone H3K36, but it is not known if Set2 regulates TC-NER. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking Set2.
2022-02-21 | GSE179794 | GEO
Project description:Nucleotide excision repair (NER) guarantees genome integrity against UV light-induced DNA damage. After UV irradiation, cells have to cope with a general transcriptional block. To ensure UV lesions repair specifically on transcribed genes, NER is coupled with transcription in an extremely organized pathway known as transcription-coupled repair. In highly metabolic cells, more than 60% of total cellular transcription results from RNA polymerase I activity. Repair of the mammalian transcribed ribosomal DNA has been scarcely studied. UV lesions severely block RNA polymerase I activity and the full transcription-coupled repair machinery corrects damage on actively transcribed ribosomal DNAs. After UV irradiation, RNA polymerase I is more bound to the ribosomal DNA and both are displaced to the nucleolar periphery. Importantly, the reentry of RNA polymerase I and the ribosomal DNA is dependent on the presence of UV lesions on DNA and independent of transcription restart.
| S-EPMC6055190 | biostudies-literature
Project description:Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.
| S-EPMC8205993 | biostudies-literature
Project description:Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.
| S-EPMC8936446 | biostudies-literature
Project description:DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.
| S-EPMC4705403 | biostudies-literature
Project description:We characterized the role of H3K36 methylation in regulating repair of UV damage from the transcribed strand (TS) of yeast genes by the transcription coupled nucleotide excision repair (TC-NER) pathway. TC-NER is triggered when RNA polymerase stalls at UV damage, such as a UV-induced cyclobutane pyrimidine dimer (CPD). During transcription, the histone methyltransferase Set2 methylates histone H3K36, but it is not known if H3K36 methylation regulates TC-NER. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells containing mutants in histone H3K36 (or set2).
2022-02-21 | GSE194101 | GEO