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