title: Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.
abstract: Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Tiago J
authors.initials: TJ
authors.lastname: Dantas
authors.forename: Owen M
authors.initials: OM
authors.lastname: Daly
authors.forename: Pauline C
authors.initials: PC
authors.lastname: Conroy
authors.forename: Martin
authors.initials: M
authors.lastname: Tomas
authors.forename: Yifan
authors.initials: Y
authors.lastname: Wang
authors.forename: Pierce
authors.initials: P
authors.lastname: Lalor
authors.forename: Peter
authors.initials: P
authors.lastname: Dockery
authors.forename: Elisa
authors.initials: E
authors.lastname: Ferrando-May
authors.forename: Ciaran G
authors.initials: CG
authors.lastname: Morrison
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D018385:Centrosome / Q000378:metabolism / Q000648:ultrastructure; D002645:Chickens; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding
chemical_list: D001324:Autoantigens; C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D008869:Microtubule-Associated Proteins; C509189:NEDD1 protein, human; C086074:PCM1 protein, human; C540807:POC5 protein, human; D049452:Green Fluorescent Proteins; C086984:XPC protein, human; D002118:Calcium