Dub3 controls DNA damage signalling by direct deubiquitination of H2AX.
A crucial event in the DNA damage response is the phosphorylation and subsequent ubiquitination of H2AX, required for the recruitment of proteins involved in DNA repair. Here we identify a novel regulator of this process, the ubiquitin hydrolase Dub3. Overexpression of wild type, but not catalytic inactive, Dub3 decreases the DNA damage-induced mono-ubiquitination of H2A(X) whereas downregulation of Dub3 has the opposite effect. Dub3 overexpression abrogates focus formation of 53BP1 and BRCA1 in response to genotoxic stress. However, focus formation of MDC1 and γH2AX, earlier events in this response, are unaffected by Dub3 overexpression. We show that Dub3 counteracts H2AX E3 ligases RNF8 and RNF168. Moreover, Dub3 and H2AX interact and Dub3 deubiquitinates H2AX in vitro. Importantly, overexpression of Dub3 delays H2AX dephosphorylation and recovery of MDC1 focus formation at later time points after DNA damage, whereas H2AX dephosphorylation at later time points is faster after Dub3 depletion. Altogether these results show that Dub3 regulates a correct DNA damage response by controlling H2AX ubiquitination.
Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n La Cuesta, La Laguna 38320, Tenerife, Spain.
M Rocío
MR
Delgado-Díaz
Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n La Cuesta, La Laguna 38320, Tenerife, Spain.
Yusé
Y
Martín
Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n La Cuesta, La Laguna 38320, Tenerife, Spain.
Anna
A
Berg
Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n La Cuesta, La Laguna 38320, Tenerife, Spain.
Raimundo
R
Freire
Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n La Cuesta, La Laguna 38320, Tenerife, Spain. Electronic address: vsmits@ull.es.
Veronique A J
VA
Smits
D019313:BRCA1 Protein / Q000378:metabolism; D002460:Cell Line; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
D019313:BRCA1 Protein; D004268:DNA-Binding Proteins; D006657:Histones; C486521:RNF8 protein, human; D025801:Ubiquitin; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C484854:USP17L2 protein, human
Deubiquitylating enzyme (Dub); H2AX; RNF168; RNF8; Ubiquitin hydrolase