Mechanisms that regulate intracellular DNA entanglement

Abstract

Topoisomerase II often produces DNA knots and catenates when its DNA strand passage activity equilibrates the topology of intracellular DNA. However, these DNA entanglements are detrimental for the normal development of genomic transactions such as replication and transcription. Fortunately, there is a mechanism actively removing these unwanted DNA entanglements in vivo. More specifically, previous studies performed in our laboratory uncovered that the in vivo correlation between knot formation and chromatin length linearly increased up until a length of 5 Kb (about 25 nucleosomes) but then reached a plateau in larger chromatin domains. This inflection is inconsistent with the expected increasing linear correlation between knot formation and chain length observed in vitro and in silico. In order to clarify which mechanism is actively minimizing the DNA entanglements in intracellular chromatin, three plausible mechanisms were proposed and tested in this thesis.

First, the possibility that in vivo DNA supercoiling could bias topoisomerase II activity towards untangling the genome was tested. Nevertheless, experimental results revealed the opposite effect. Accumulation of positive DNA supercoiling during transcription increases the DNA’s knotting probability 25-fold. Second, the assumption that topoisomerase II alone is capable of minimizing the overall DNA entanglement to values below the thermodynamic equilibrium was tested. The experimental results indicate that even though this ability is functioning in vitro, it does not minimize the overall entanglement of intracellular chromatin. Finally, the possibility that the loop extrusion capacity of SMC (structural maintenance of chromosomes) complexes, such as condensin and cohesin could help to disentangle the genome was also tested. The loop extrusion process could tighten DNA entanglements towards the outside of the loops and consequently enforce their removal by topoisomerase II. These experiments uncovered that the activity of condensin, but not of cohesin, promotes the resolution of DNA knots formed within chromatin fibers both in interphase and mitosis. Moreover, inactivation of condensin restores the expected linear correlation between DNA knot formation and chromatin length. Condensin is very well known for its role during mitosis, however, its role during interphase remained mostly unknown. The results of this thesis suggest that condensin is able to extrude DNA loops in order to minimize DNA entanglements throughout the entire cell cycle. This critical role could explain why inactivation of condensin during interphase is followed by many genome dysfunctions.