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.
Type 2 topoisomerases, in particular the alpha isoform in human cells, play a key role in cohesion and sister chromatid separation during mitosis. These enzymes are thus vital for cycling cells and are obvious targets in cancer chemotherapy. Evidence obtained in yeast and Xenopus model systems indicates that conjugation of topoisomerase 2 with small ubiquitin-like modifier (SUMO) proteins is required for its mitotic functions. Here, we provide biochemical and cytologic evidence that topoisomerase 2 alpha is conjugated to SUMO-2/3 during interphase and mitosis in response to topoisomerase 2 inhibitors and "poisons" (ICRF-187, etoposide, doxorubicin) that stabilize catalytic intermediates (cleavage complexes, closed clamp forms) of the enzyme onto target DNA. During mitosis, SUMO-2/3-modified forms of topoisomerase 2 alpha localize to centromeres and chromosome cores/axes. However, centromeres are unresponsive to inhibitors during interphase. Furthermore, formation of topoisomerase 2 alpha-SUMO-2/3 conjugates within mitotic chromosomes strongly correlates with incomplete chromatid decatenation and decreases progressively as cells approach the metaphase-anaphase transition. We also found that the PIASy protein, an E3 ligase for SUMO proteins, colocalizes with SUMO-2/3 at the mitotic chromosomal cores/axes and is necessary for both formation of SUMO-2/3 conjugates and proper chromatid segregation. We suggest that the efficacy of topoisomerase inhibitors to arrest cells traversing mitosis may relate to their targeting of topoisomerase 2 alpha-SUMO-2/3 conjugates that concentrate at mitotic chromosome axes and are directly involved in chromatid arm separation.
Agostinho
Marta
M
Instituto de Medicina Molecular, Faculdade de Medicina, Lisboa, Portugal.
Santos
Vera
V
Ferreira
Fernando
F
Costa
Rafael
R
Cardoso
Joana
J
Pinheiro
Inês
I
Rino
José
J
Jaffray
Ellis
E
Hay
Ronald T
RT
Ferreira
João
J
D000970:Antineoplastic Agents / Q000494:pharmacology; D002384:Catalysis; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002877:Chromosomes, Human / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008938:Mitosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism; D011929:Razoxane / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D059005:Topoisomerase II Inhibitors
D000970:Antineoplastic Agents; D002843:Chromatin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C485891:PIAS4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D059005:Topoisomerase II Inhibitors; D011929:Razoxane; D005047:Etoposide; D004250:DNA Topoisomerases, Type II