TY - JOUR
T1 - A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest
AU - Deiss, Katharina
AU - Lockwood, Nicola
AU - Howell, Michael
AU - Segeren, Hendrika Alida
AU - Saunders, Rebecca E.
AU - Chakravarty, Probir
AU - Soliman, Tanya N.
AU - Martini, Silvia
AU - Rocha, Nuno
AU - Semple, Robert
AU - Zalmas, Lykourgos Panagiotis
AU - Parker, Peter J.
PY - 2019/4/8
Y1 - 2019/4/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85064483377&partnerID=8YFLogxK
U2 - 10.1093/nar/gky1295
DO - 10.1093/nar/gky1295
M3 - Article
C2 - 30590722
AN - SCOPUS:85064483377
SN - 1362-4962
VL - 47
SP - 2906
EP - 2921
JO - Nucleic acids research
JF - Nucleic acids research
IS - 6
ER -
