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A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest
journal contributionposted on 2019-12-12, 17:34 authored by Katharina Deiss, Nicola Lockwood, Michael Howell, Hendrika Alida Segeren, Rebecca E Saunders, Probir Chakravarty, Tanya N Soliman, Silvia Martini, Nuno Rocha, Robert Semple, Lykourgos-Panagiotis Zalmas, Peter J Parker
The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest.
Cell Cycle ProteinsCell LineChromosomal Proteins, Non-HistoneDNA DamageDNA Topoisomerases, Type IIFibroblastsG2 Phase Cell Cycle CheckpointsGenome, HumanGerm-Line MutationHumansLigasesMultiprotein ComplexesPiperazinesPoly-ADP-Ribose Binding ProteinsRNA InterferenceSumoylationUbiquitin-Protein LigasesParker FC001130Swanton FC001169HTSCBPC-ackFC-ackCS-ackLM-ackDevelopmental Biology05 Environmental Sciences06 Biological Sciences08 Information and Computing Sciences