RESUMO
Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.
Assuntos
DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Ubiquitina/metabolismo , Sequência de Aminoácidos , Linhagem Celular/efeitos da radiação , Núcleo Celular/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Tetra-Hidrofolato Desidrogenase/genética , Ubiquitina/genética , Raios UltravioletaRESUMO
Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.