Impact of DNA Topology on cellular metabolism: in vivo and in vitro approaches to study chromatin dynamics and DNA Topoisomerases.

Abstract

DNA topology describes the tertiary conformations of DNA and it influences all the fundamental biological processes such as transcription, replication and recombination. DNA topoisomerases are essential enzymes able to covalently modify DNA topology and they are emerging as important factors in a wide range of fundamental metabolic processes in both the nuclear and mitochondrial genomes. Furthermore the study of topoisomerases is therapeutically relevant to cancer, immune disorders and neurological diseases. Topoisomerases introduce transient DNA breaks using a trans-esterification mechanism, which is highly reversible and minimizes the risks to genome stability that would otherwise occur owing to strand breakage. Furthermore the topological state of DNA is not equal among different classes of organisms. In mesophilic organisms DNA is negatively supercoiled while positive supercoiling is a peculiar mark of thermophilic organisms living at high temperatures. During my PhD I analyzed different aspects related to DNA topology, in particular: • the relationship between DNA topology and transcription was investigated. DNA supercoiling has been shown to be essential for transcriptional regulation. My analysis demonstrates that DNA topology is a determinant of the selection of transcription starting site. • the structural diversity of negatively and positively supercoiled DNA molecules was characterized by biochemical assays and atomic force microscopy. My data demonstrate that supercoiling strongly influences DNA structure. In particular the positive supercoiling is more stable than negative protecting DNA from nuclease degradation and helix breathing. These data support the potential role of positive supercoiling in DNA stability maintenance. • the biochemical properties of topoisomerases inhibitor Daunomycin and its derivative were investigated. It’s known that human topoisomerases are important target of several antitumor agents such as anthracyclines. However their use in chemoterapy is limited by a lot of side effects especially cardiotoxicity. One approach to overcome this problem consists in testing new molecular transporters (such as oligopeptides) that do not alter the characteristics of the drugs but reduce the side effects. My results demonstrate that Daunomycin conjugated with an oligopeptide containing six arginines retains the same biochemical properties of the free drug, representing a good alternative to Daunomycin. • I described the in vivo detection of reverse gyrase by using a thermostable protein-tag in the thermophilic organism Sulfolobus islandicus. Reverse gyrase is a DNA topoisomerase with the unique capability to introduce positive supercoils in DNA. Although a lot of in vitro studies have been performed to understand the mechanical properties of positive supercoiling reaction, its biological role in vivo remains unclear. This new thermostable protein-tag will be useful for reverse gyrase analysis both in vitro and in vivo.