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Nucleic Acids Res. 2019 Apr 8;47(6):2906-2921. doi: 10.1093/nar/gky1295.

A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest.

Author information

1
Protein Phosphorylation Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
2
High Throughput Screening, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
3
Bioinformatics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
4
The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge CB2 0QQ, UK.
5
The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, UK.
6
Translational Cancer Therapeutics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
7
School of Cancer and Pharmaceutical Sciences King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

Abstract

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.

PMID:
30590722
PMCID:
PMC6451093
DOI:
10.1093/nar/gky1295
[Indexed for MEDLINE]
Free PMC Article

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