A genome-wide RNAi screen identifies the SMC 5 / 6 complex as a non-redundant regulator of a Topo 2 a-dependent G 2 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 counterscreened 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.

[1]  Topoisomerase II , 2020, Definitions.

[2]  Mariana Díaz,et al.  Scaffolding for Repair: Understanding Molecular Functions of the SMC5/6 Complex , 2018, Genes.

[3]  Marius A. Micluţa,et al.  Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIβ in regulation of the decatenation checkpoint , 2017, Nucleic acids research.

[4]  P. Sicinski,et al.  Cell cycle proteins as promising targets in cancer therapy , 2017, Nature Reviews Cancer.

[5]  R. Hoebe,et al.  Non-SMC Element 2 (NSMCE2) of the SMC5/6 Complex Helps to Resolve Topological Stress , 2016, International journal of molecular sciences.

[6]  G. Stewart,et al.  RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation , 2016, Nature Communications.

[7]  P. Jordan,et al.  Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression , 2016, Development.

[8]  F. Uhlmann.  SMC complexes: from DNA to chromosomes , 2016, Nature Reviews Molecular Cell Biology.

[9]  O. Fernandez-Capetillo.  The (elusive) role of the SMC5/6 complex , 2016, Cell cycle.

[10]  A. Lengronne,et al.  Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance , 2015, Molecular cell.

[11]  Y. Liu,et al.  PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis , 2015, Nature Communications.

[12]  M. Blasco,et al.  NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity , 2015, The EMBO journal.

[13]  C. Sjögren,et al.  The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker. , 2015, Cell reports.

[14]  L. Collinson,et al.  Mitotic catenation is monitored and resolved by a PKCε-regulated pathway , 2014, Nature Communications.

[15]  Ryuichiro Nakato,et al.  The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement , 2014, PLoS genetics.

[16]  S. O’Rahilly,et al.  Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance. , 2014, The Journal of clinical investigation.

[17]  S. Pavey,et al.  Decatenation checkpoint‐defective melanomas are dependent on PI3K for survival , 2014, Pigment cell & melanoma research.

[18]  K. Shirahige,et al.  Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells , 2014, Molecular biology of the cell.

[19]  S. Pavey,et al.  Defective decatenation checkpoint function is a common feature of melanoma. , 2014, The Journal of investigative dermatology.

[20]  D. J. Clarke,et al.  Direct Monitoring of the Strand Passage Reaction of DNA Topoisomerase II Triggers Checkpoint Activation , 2013, PLoS genetics.

[21]  M. Roizen,et al.  Hallmarks of Cancer: The Next Generation , 2012 .

[22]  T. Itoh,et al.  Chromosome length influences replication-induced topological stress , 2011, Nature.

[23]  S. Nagai,et al.  Nuclear organization in genome stability: SUMO connections , 2011, Cell Research.

[24]  R. Paules,et al.  Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM , 2010, Cell cycle.

[25]  K. Laukens,et al.  A manually curated network of the PML nuclear body interactome reveals an important role for PML-NBs in SUMOylation dynamics , 2010, International journal of biological sciences.

[26]  A. Kegel,et al.  The Smc5/6 complex: more than repair? , 2010, Cold Spring Harbor symposia on quantitative biology.

[27]  A. Irmisch,et al.  Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes , 2009, Molecular and Cellular Biology.

[28]  J. Bachant,et al.  SUMO modification of DNA topoisomerase II: trying to get a CENse of it all. , 2009, DNA repair.

[29]  Junjie Chen,et al.  Topoisomerase IIα controls the decatenation checkpoint , 2009, Nature Cell Biology.

[30]  K. Shuai,et al.  Resolution of Sister Centromeres Requires RanBP2-Mediated SUMOylation of Topoisomerase IIα , 2008, Cell.

[31]  R. Hay,et al.  Conjugation of human topoisomerase 2 alpha with small ubiquitin-like modifiers 2/3 in response to topoisomerase inhibitors: cell cycle stage and chromosome domain specificity. , 2008, Cancer research.

[32]  A. Carr,et al.  Smc5/6: a link between DNA repair and unidirectional replication? , 2008, Nature Reviews Molecular Cell Biology.

[33]  M. Speicher,et al.  Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation , 2007, Chromosoma.

[34]  Hongtao Yu,et al.  The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins , 2007, Nature Structural &Molecular Biology.

[35]  D. Gillespie,et al.  Chk1 is required for G2/M Checkpoint Response Induced by the Catalytic Topoisomerase II Inhibitor ICRF-193 , 2007, Cell cycle.

[36]  A. Sharrocks,et al.  An extended consensus motif enhances the specificity of substrate modification by SUMO , 2006, The EMBO journal.

[37]  Hongtao Yu,et al.  Human MMS21/NSE2 Is a SUMO Ligase Required for DNA Repair , 2005, Molecular and Cellular Biology.

[38]  Junjie Chen,et al.  BRCA1 participates in DNA decatenation , 2005, Nature Structural &Molecular Biology.

[39]  T. Anan,et al.  PIASy mediates SUMO‐2 conjugation of Topoisomerase‐II on mitotic chromosomes , 2005, The EMBO journal.

[40]  A. Lehmann.  The role of SMC proteins in the responses to DNA damage. , 2005, DNA repair.

[41]  Graham Dellaire,et al.  PML nuclear bodies: dynamic sensors of DNA damage and cellular stress , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[42]  S. Müller,et al.  SUMO: a regulator of gene expression and genome integrity , 2004, Oncogene.

[43]  T. Kitajima,et al.  The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis , 2004, Nature.

[44]  中川 拓.  Identification of decatenation G2 checkpoint impairment independently of DNA damage G2 checkpoint in human lung cancer cell lines , 2004 .

[45]  James J. Yoo,et al.  Cell cycle checkpoint function in bladder cancer. , 2003, Journal of the National Cancer Institute.

[46]  M. Dasso,et al.  SUMO-2/3 regulates topoisomerase II in mitosis , 2003, The Journal of cell biology.

[47]  A. Escargueil,et al.  Catalytic topoisomerase II inhibitors in cancer therapy. , 2003, Pharmacology & therapeutics.

[48]  J. Oshima,et al.  The G2-phase decatenation checkpoint is defective in Werner syndrome cells. , 2003, Cancer research.

[49]  S. Elledge,et al.  The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II. , 2002, Molecular cell.

[50]  A. Dejean,et al.  SUMO: of branched proteins and nuclear bodies , 2001, Oncogene.

[51]  Stephen J. Elledge,et al.  Cell Cycle Checkpoints: Preventing an Identity Crisis , 1996, Science.

[52]  D. J. Clarke,et al.  A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells , 1994, Nature.

[53]  D. J. Clarke,et al.  Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks. , 1993, Journal of cell science.

[54]  Leland Hartwell,et al.  Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells , 1992, Cell.

[55]  F. M. Davis,et al.  Monoclonal antibodies to mitotic cells. , 1983, Proceedings of the National Academy of Sciences of the United States of America.