Deiss, Katharina Lockwood, Nicola Howell, Michael Segeren, Hendrika Alida Saunders, Rebecca E Chakravarty, Probir Soliman, Tanya N Martini, Silvia Rocha, Nuno Semple, Robert Zalmas, Lykourgos-Panagiotis Parker, Peter J A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest 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 Proteins;Cell Line;Chromosomal Proteins, Non-Histone;DNA Damage;DNA Topoisomerases, Type II;Fibroblasts;G2 Phase Cell Cycle Checkpoints;Genome, Human;Germ-Line Mutation;Humans;Ligases;Multiprotein Complexes;Piperazines;Poly-ADP-Ribose Binding Proteins;RNA Interference;Sumoylation;Ubiquitin-Protein Ligases;Parker FC001130;Swanton FC001169;HTS;CB;PC-ack;FC-ack;CS-ack;LM-ack;Developmental Biology;05 Environmental Sciences;06 Biological Sciences;08 Information and Computing Sciences 2019-12-12
    https://crick.figshare.com/articles/journal_contribution/A_genome-wide_RNAi_screen_identifies_the_SMC5_6_complex_as_a_non-redundant_regulator_of_a_Topo2a-dependent_G2_arrest/11357801