RESUMO
Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.
Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Adutos de DNA/metabolismo , Adutos de DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo por Excisão , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição , Transcrição Gênica , Ubiquitinação , Raios UltravioletaRESUMO
Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.
Assuntos
Bacillus subtilis , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ribossomos/metabolismo , Peptídeos/metabolismoRESUMO
Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains and reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation by the ribosome quality control pathway. Notably, we see no evidence of mRNA cleavage by MutS2, nor does it promote ribosome rescue by tmRNA as SmrB cleavage does in E. coli. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in various bacteria.
