RESUMEN
Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.
Asunto(s)
Microscopía por Crioelectrón , Reparación del ADN , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Transcripción Genética , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Humanos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/ultraestructura , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Transcripción/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , UbiquitinaciónRESUMEN
The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.
Asunto(s)
ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Elonguina/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Polimerasa II/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Síndrome de Cockayne/enzimología , Síndrome de Cockayne/genética , ADN Helicasas/química , ADN Helicasas/ultraestructura , Reparación del ADN/genética , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/ultraestructura , Elonguina/química , Elonguina/ultraestructura , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/ultraestructura , ARN Polimerasa II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/ultraestructura , Ubiquitinación/genéticaRESUMEN
The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.
