RESUMO
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Assuntos
DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , Proteólise , RNA Polimerase II , Transcrição Gênica , Humanos , Proteínas de Transporte , DNA/metabolismo , DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.
Assuntos
Caenorhabditis elegans , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Fator de Transcrição TFIIH , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Animais , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/metabolismo , Endonucleases/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Mutação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that protects against the negative effects of transcription-blocking DNA lesions. Hereditary TC-NER deficiencies cause pleiotropic and often severe neurodegenerative and progeroid symptoms. While multiple assays have been developed to determine TC-NER activity for clinical and research purposes, monitoring TC-NER is hampered by the low frequency of repair events occurring in transcribed DNA. 'Recovery of RNA Synthesis' is widely used as indirect TC-NER assay based on the notion that lesion-blocked transcription only resumes after successful TC-NER. Here, we show that measuring novel synthesis of a protein after its compound-induced degradation prior to DNA damage induction is an equally effective but more versatile manner to indirectly monitor DNA repair activity in transcribed genes. This 'Recovery of Protein Synthesis' (RPS) assay can be adapted to various degradable proteins and readouts, including imaging and immunoblotting. Moreover, RPS allows real-time monitoring of TC-NER activity in various living cells types and even in differentiated tissues of living organisms. To illustrate its utility, we show that DNA repair in transcribed genes declines in aging muscle tissue of C. elegans. Therefore, the RPS assay constitutes an important novel clinical and research tool to investigate transcription-coupled DNA repair.
Assuntos
Caenorhabditis elegans , Reparo do DNA , Biossíntese de Proteínas , Transcrição Gênica , Animais , Caenorhabditis elegans/fisiologia , DNA/metabolismo , Dano ao DNA , Envelhecimento/metabolismo , Músculos/metabolismoRESUMO
Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.
Assuntos
Cromatina , Inibidores de Poli(ADP-Ribose) Polimerases , Cromatina/genética , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismoRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Reparo do DNA/genética , DNA de Helmintos/fisiologia , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) protect cells against a variety of helix-distorting DNA lesions. In C. elegans, GG-NER primarily acts in proliferative germ cells and embryos, while TC-NER acts in post-mitotic somatic cells to maintain transcription. We leverage this difference to distinguish whether proteins function in GG-NER and/or TC-NER by straightforward UV survival assays. Here, we detail a protocol for these assays, using GG-NER factor xpc-1 and TC-NER factor csb-1 as examples. For complete details on the use and execution of this protocol, please refer to Sabatella et al. (2021).
Assuntos
Caenorhabditis elegans/fisiologia , Reparo do DNA , Transcrição Gênica , AnimaisRESUMO
Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.
Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , Fator 1 de Elongação de Peptídeos/metabolismo , Elongação da Transcrição Genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Fator 1 de Elongação de Peptídeos/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , UbiquitinaçãoRESUMO
Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.