DNA Repair 11 (2012) 892–905 Contents lists available at SciVerse ScienceDirect DNA Repair j ourna l ho me pag e: www.elsevier.com/locate/dnarepair RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells Vibe H. Oestergaard a , Constanze Pentzold a , Rune Troelsgaard Pedersen a , Silviu Iosif a , Arno Alpi b , Simon Bekker-Jensen c , Niels Mailand c , Michael Lisby a,∗ a Department of Biology, University of Copenhagen, DK-2200, Copenhagen N, Denmark b The Scottish Institute for Cell Signalling, University of Dundee, Dundee, United Kingdom c Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200, Copenhagen N, Denmark a r t i c l e i n f o Article history: Received 13 April 2012 Received in revised form 2 August 2012 Accepted 27 August 2012 Available online 24 September 2012 Keywords: HERC2 RNF8 RNF168 Ubiquitin DT40 a b s t r a c t The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double- strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Generation of DNA double-strand breaks (DSBs) triggers a cas- cade of events in the surrounding chromatin initiated by ATM- and DNA-PK-mediated phosphorylation of histone H2AX (-H2AX), which is subsequently maintained by ATR (reviewed in [1–3]). In human cells, -H2AX formation quickly attracts a number of proteins including MDC1 [4], which in turn recruits the E3 ubiquitin ligase RNF8 in a phosphorylation-dependent manner [5–7]. RNF8 initiates chromatin ubiquitylation and thereby engages another E3 ubiquitin ligase RNF168, which is recruited through its ubiquitin interacting domain [8,9]. RNF8 and RNF168 sub- sequently catalyze polyubiquitylation of the chromatin flanking the DSB, thereby triggering ubiquitin-dependent recruitment of a second class of proteins [5–9]. The latter class of proteins includes 53BP1, which promotes non-homologous end-joining (NHEJ) [10,11] and BRCA1, which facilitates recombinational repair [12,13]. Recently, another E3 ubiquitin ligase, HERC2, was found to be a part of the DNA damage-induced ubiquitin-signaling cascade [14]. Studies conducted in human cells show that HERC2 inter- acts directly with RNF8 upon DNA damage. The mechanistic basis for the RNF8–HERC2 interaction involves DNA damage-dependent ∗ Corresponding author. Tel.: +45 3532 2120; fax: +45 3532 2128. E-mail address: mlisby@bio.ku.dk (M. Lisby). phosphorylation of human HERC2 on T4827. HERC2 is a HECT domain E3 ubiquitin ligase, which counts almost 5000 amino acids. The extraordinary size of HERC2 is likely the reason why full-length HERC2 cDNA has not been isolated, and consequently, the function of particular amino acids has not been addressed hitherto. In par- ticular, it has not yet been investigated whether HERC2 employs its own ubiquitin ligase activity in the DSB-associated chromatin response. Other recent studies of HERC2 indicate that it can func- tion as an E3 ubiquitin ligase toward BRCA1 and XPA [15–17]. The HERC2-mediated polyubiquitylation of BRCA1 and XPA has been suggested to target these substrates for proteasomal degradation. Studies of RNF8 and RNF168 knockout mice have confirmed the importance of these E3 ubiquitin ligases in the DNA damage- induced ubiquitylation cascade [18–20]. In addition, RNF168 mutations have been linked to the human RIDDLE syndrome, whose clinical features are an increased radiosensitivity, immunodefi- ciency, dysmorphic features, and learning difficulties [9] and as such solid genetic systems for exploring the functions of RNF8 and RNF168 exist. In contrast until now the involvement of HERC2 in the DNA damage response and its relation to RNF8 and RNF168 have not been studied in a genetically tractable system. Here we present a genetic analysis of the three E3 ubiquitin lig- ases RNF8, RNF168 and HERC2 using the vertebrate cell line DT40. Our results confirm that RNF8 and RNF168 are crucial for DNA damage-induced ubiquitylation and for the cell survival after DNA damage. Surprisingly, we find that despite remarkable conservation 1568-7864/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2012.08.005