Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes

Jessel Ayra-Plasencia,Oliver Quevedo,Félix Machín,Silvia Santana-Sosa,Marcos Zamora-Dorta,Grant W Brown,Emiliano Matos-Perdomo,Michael Lisby,Cristina Ramos-Pérez,Sara Medina-Suárez

doi:10.1038/s41598-021-93875-5

Abstract

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.

Highlights

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids
The overall DNA content we quantified by FACS, as well as the absence of ingel smears and broken ribosomal DNA array (rDNA) in the Southern blots, strongly suggests that the disappearance of bands correlates with entrapment of chromosomes in the loading well (Figs. 1, 2, 5, S4–S6)
We distinguished two classes of chromosomes affected by the PulsedField Gel Electrophoresis (PFGE) retention: the rDNA-bearing chromosome XII and all the other 15 chromosomes

Summary

Introduction

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. We here show that top[2,3,4,5] chromosomes fail to enter a PulsedField Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. During DNA replication, cells check that the replication fork (RF) does not get stalled or blocked, or that long stretches of single-stranded DNA (ssDNA) are not left behind the RF, yet cells can enter anaphase with unfinished replication if it proceeds too slowly compared to the cell division rate[1,4,5] Likewise, cells monitor both DNA double-strand breaks (DSBs) and ssDNA during DNA damage and early steps of its repair through the homologous recombination (HR) pathway, but not the direct presence of recombination intermediates that connect the damaged DNA with its sister template[6,7,8]. We show that Top[2] actions must take place at the correct time, after which the change in chromosome structure becomes refractory to Top[2] activity

Methods

Results

Conclusion