Novel mitotic DNA topology regulators

The double helical structure of DNA imposes a major challenge to all processes involving this molecule: access to its sequence. This requires DNA unwinding, which creates massive changes in the topology of DNA, inducing helical torsion, supercoiling and ultimately sister chromatid intertwining. These topological changes are problematic and need to be resolved to ensure genome stability. Topoisomerases are the key enzymes capable of solving these topological issues and hence critical to all processes involving DNA (Pommier et al. 2016). Topoisomerases operate throughout the entire cell cycle. Yet, their action during mitosis offers the last chance to resolve DNA entanglements, a key requisite for proper genome resolution and segregation. However, how mitotic chromosome resolution is executed is not fully understood. Uncovering this critical process is key to understand how chromosome organization is regulated during this important cell cycle stage.
Additionally, genome resolution mechanisms can unravel novel potential sources for genome instability, and their potential impact on disease development and treatment. Recent work from our lab highlighted that DNA topology is dynamically maintained throughout mitosis by balancing DNA dis)entanglements (Piskadlo, Tavares, and Oliveira 2017). We showed that the major enzyme that removes sister chromatid linkages (Topoisomerase 2, Top2) is a bidirectional enzyme, able to resolve DNA linkages but also re-introduce DNA entanglements (Piskadlo and Oliveira 2017, Piskadlo, Tavares, and Oliveira 2017). Yet, it is not fully understood how this bidirectional reaction is significantly biased towards the resolution of chromatid intertwines (and thereby ensures chromatid individualization). Failures in these topology-resolution bias mechanisms have drastic consequences on cells, as they would enable re-introduction of DNA catenations and thereby compromise genome segregation. Our findings therefore raise the possibility that regulation of DNA topology throughout mitosis requires a stricter control than previously anticipated. This proposal aims to uncover novel topology regulators that cooperate in this process, in order to achieve timely and efficient genome partition at every cell division.
We will combine classical genetic approaches with advanced imaging technologies to identify the role of novel topology regulators during mitosis, and their molecular mechanisms. We and others have recently uncovered that Structural Maintenance of Chromosome (SMC) complexes, particularly cohesin and condensin, are major players in determining the extent and direction of Top2 towards catenation/recatenation of sister DNA molecules (see extended discussion in (Piskadlo and Oliveira 2017)). These interactions, therefore, offer a unique tool to screen for novel topology regulators: knock-down of a putative topology regulator should give rise to mild catenation defects. These defects should be able to rescue cohesion problems and exacerbate condensin defects. Previous studies in our lab, in collaboration with Dr. Rui Silva (Univ. Algarve, co-PI) and Dr. Rui Martinho (Univ. Aveiro, consultant), have successfully applied unbiased genetic screens in Drosophila wings to identify modulators of sister chromatid cohesion (Silva et al. 2018). Unpublished observations from our laboratory revealed that several of the identified suppressors are essential for proper sister chromatid resolution during mitosis, as evidenced by the high degree of chromatin bridges observed when these factors are depleted. We therefore aim to exploit the role of these and search for other putative DNA topology modulators in the process of sister chromatid resolution during mitosis. This will be performed by combining quantitative live cell imaging analysis, coupled with a unique technology for acute protein inactivation, pioneered in our lab (Carvalhal et al. 2018, Mirkovic et al. 2015, Oliveira et al. 2010, Piskadlo, Tavares, and Oliveira 2017), to dissect the role of these players in mitosis with unprecedented temporal resolution. We will additionally uncover the molecular mechanisms of identified players, using advanced biochemical approaches, in collaboration with Prof. Ian Hickson, Univ. Copenhagen.
Uncovering novel players in this essential process holds the prospect of identifying novel routes to genome instability that may underly human pathologies associated with chromosome mis-segregation (e.g. cancer, infertility, development disorders). Additionally, these studies offer the potential of identifying novel therapeutic targets for cancer treatment. Several topoisomerase inhibitors are indeed widely used as genotoxic agents in cancer treatment, although accompanied by elevated toxicity (Liang et al. 2019, Skok et al. 2020). We anticipate that mitosis-specific regulation of DNA topology holds the prospect of offering more selective therapeutic approaches to target such a wide-spread disease.