Project : Impact of blocked Top1 cleavage complexes on genome stability during the cell cycle

The integrity of the genetic information stored in DNA is constantly challenged by various types of DNA lesions. DNA topoisomerase I (Top1) has been involved in generating deleterious genomic lesions associated with irreversible topoisomerase cleavage complexes (Top1ccs), in which the enzyme remains covalently bound to one end of a single-strand DNA break. It has been shown recently that these lesions occur naturally at higher frequencies than previously thought, leading to increased mutagenesis and chromosomal rearrangements. Hence, blocked Top1ccs greatly impact genome stability and could trigger various genetic diseases. Despite intense research on the topic, our understanding on the impact of blocked Top1ccs during the cell cycle is still limited. On one hand, Top1ccs have been shown to affect gene transcription in cells in G₀/G₁ phase of the cell cycle. On the other hand, the majority of the DNA lesions induced by Top1ccs, DNA double-strand breaks (DSBs), occurs during DNA replication in S phase. DSBs most likely originate from the conflicts between the transcription and the replication machineries but it has never been formally demonstrated and how DSBs are produced is still unclear. In this project, we aim to characterize the consequences of blocked Top1ccs on transcription and replication by combining complementary genome-wide approaches using next-generation sequencing and a site-specific approach using fluorescence microscopy. We will use the yeast cellular model Saccharomyces cerevisiae, whose powerful genetics and molecular biology are suitable to achieve rapidly the project’s objectives.