gkw216.pdf (3.71 MB)
A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair
journal contributionposted on 2020-09-14, 10:35 authored by Michael Ranes, Stefan Boeing, Yuming Wang, Franziska Wienholz, Hervé Menoni, Jane Walker, Vesela Encheva, Probir Chakravarty, Pierre-Olivier Mari, Aengus Stewart, Giuseppina Giglia-Mari, Ambrosius P Snijders, Wim Vermeulen, Jesper Q Svejstrup
Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.
Amino Acid SequenceCell CycleCell LineCell SurvivalCluster AnalysisCockayne SyndromeDNA DamageDNA RepairGene ExpressionGene Expression ProfilingGenomic InstabilityHumansMutationOxidative StressRecombinant Fusion ProteinsTranscription, GeneticUbiquitinationSvejstrup FC001166CBPRTCS-ack05 Environmental Sciences06 Biological Sciences08 Information and Computing SciencesDevelopmental Biology