A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest 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 10779/crick.11357801.v1 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 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. 2019-12-12 17:34:32 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