Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair.
Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumorigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2 enzymes for impacts on cellular DSB responses. With a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3 ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Christine K
CK
Schmidt
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Yaron
Y
Galanty
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Matylda
M
Sczaniecka-Clift
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Julia
J
Coates
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Satpal
S
Jhujh
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Mukerrem
M
Demir
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.
Matthew
M
Cornwell
Institute of Molecular Biology (IMB), 55128 Mainz, Germany.
Petra
P
Beli
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, UK.|The Wellcome Trust Sanger Institute, Hinxton, CB10 1SA Cambridge, UK.
Stephen P
SP
Jackson
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004706:Endodeoxyribonucleases; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D059767:Recombinational DNA Repair; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D002352:Carrier Proteins; D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins; C097308:UBE2D1 protein, human; C097309:UBE2D2 protein, human; C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C577594:RNF138 protein, human; D044767:Ubiquitin-Protein Ligases; D004706:Endodeoxyribonucleases; C410741:RBBP8 protein, human