title: A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.
abstract: 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.
authors.affiliation: Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Ranes
authors.affiliation: Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Boeing
authors.affiliation: Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wienholz
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
authors.forename: Hervé
authors.initials: H
authors.lastname: Menoni
authors.affiliation: Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Walker
authors.affiliation: Protein Analysis and Proteomics Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Vesela
authors.initials: V
authors.lastname: Encheva
authors.affiliation: Bioinformatics & Biostatistics Laboratory, The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Probir
authors.initials: P
authors.lastname: Chakravarty
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, F-31077 Toulouse, France.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Mari
authors.affiliation: Bioinformatics & Biostatistics Laboratory, The Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Aengus
authors.initials: A
authors.lastname: Stewart
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, F-31077 Toulouse, France.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Giglia-Mari
authors.affiliation: Protein Analysis and Proteomics Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.affiliation: Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK jesper.svejstrup@crick.ac.uk.
authors.forename: Jesper Q
authors.initials: JQ
authors.lastname: Svejstrup
mesh_terms: D000595:Amino Acid Sequence; D002453:Cell Cycle; D002460:Cell Line; D002470:Cell Survival; D016000:Cluster Analysis; D003057:Cockayne Syndrome / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D015870:Gene Expression; D020869:Gene Expression Profiling; D042822:Genomic Instability; D006801:Humans; D009154:Mutation; D018384:Oxidative Stress; D011993:Recombinant Fusion Proteins; D014158:Transcription, Genetic; D054875:Ubiquitination
chemical_list: D011993:Recombinant Fusion Proteins