Abstract
The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.
Highlights
Type-2A topoisomerases, such as bacterial topo IV and eukaryotic topo II, pass one segment of duplex DNA through the transient double-stranded DNA break that they produce in another segment (Wang, 1998)
We used two experimental approaches to assess whether the capacity of topo II to simplify the equilibrium topology of DNA was sustaining the low knotting probability (Pkn) of intracellular chromatin
To verify that the simplification capacity of yeast topo II was targeted by ICRF-193, we added to crude lysates of the cells a negatively supercoiled DNA plasmid (YEp24, 7.8 Kb), which served as internal control of topo II activity (Fig 2A)
Summary
Type-2A topoisomerases, such as bacterial topo IV and eukaryotic topo II, pass one segment of duplex DNA through the transient double-stranded DNA break that they produce in another segment (Wang, 1998) This DNA-passage activity is essential to remove the intertwines generated between newly replicated DNA molecules and to modulate DNA supercoiling during genome transactions (Corbett & Berger, 2004; Nitiss, 2009). Computer simulations have predicted that DNA molecules confined in biological systems would be massively entangled if type-2A topoisomerases could freely equilibrate their global topology (Arsuaga et al, 2002; Micheletti et al, 2008; Dorier & Stasiak, 2009) This prospect does not occur because the hierarchical folding of chromatin, which has a scaling behavior similar to that of a fractal globule, drastically reduces the topological complexity of chromosomes (Lieberman-Aiden et al, 2009; Mirny, 2011). Reconstruction of 3D paths of high-order chromatin fibers in individual cells evidenced the scarcity of long-range entanglements (Siebert et al, 2017, Stevens et al, 2017; Sulkowska et al, 2018)
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