title: The ubiquitin ligases RNF8 and RNF168 display rapid but distinct dynamics at DNA repair foci in living cells.
abstract: Rapid assembly of DNA damage response (DDR) proteins at nuclear "repair" foci is a hallmark response of ionizing radiation (IR)-treated cells. The ubiquitin E3 ligases RNF8 and RNF168 are critical for foci formation, and here we aim to determine their dynamic mobility and abundance at individual foci in living cells. To this end, YFP-tagged RNF8 and RNF168 were expressed at physiological levels in MCF-7 cells, then analyzed by fluorescence recovery after photobleaching (FRAP) assays, nuclear retention measurement, and virus-like particles (VLPs)-based quantification. The results showed that RNF8 and RNF168 were both highly dynamic at IR-induced foci. Intriguingly, RNF8 displayed remarkably faster in vivo association/dissociation rates than RNF168, and RNF8-positive IR-foci were less resistant to detergent extraction. In addition, copy number assay revealed that RNF168 was two-fold more abundant than RNF8 at foci. Collectively, we show for the first time that RNF8 moves on-and-off nuclear DNA repair foci more than six-fold as quickly as RNF168. The faster kinetics of RNF8 recruitment explains why RNF8 is generally observed at DNA-breaks prior to RNF168. Moreover, our finding that RNF8 is less abundant than RNF168 identifies RNF8 as a rate-limiting determinant of focal repair complex assembly.
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. Electronic address: mythmok@mythmok.com.
authors.forename: Myth T S
authors.initials: MT
authors.lastname: Mok
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong; State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Shatin, NT, Hong Kong; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
authors.forename: Alfred S L
authors.initials: AS
authors.lastname: Cheng
authors.affiliation: Centre for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia.
authors.forename: Beric R
authors.initials: BR
authors.lastname: Henderson
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D057809:HEK293 Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D061986:MCF-7 Cells; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C486521:RNF8 protein, human; D025801:Ubiquitin; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases
keywords: DNA damage; Dynamics; Nuclear foci; RNF168; RNF8