Chromatin and Topological Dynamics during the Cell Cycle

DNA topoisomerases, Top1 and Top2, together with the high mobility group protein Hmo1, assist DNA replication and transcription to avoid topological stress and ensure genome stability. Hmo1 and Top2 contribute to the architectural state of transcription by stabilizing cruciform structures and protecting negative supercoils that are formed in gene boundaries in S phase. While the topological state of the genome has been characterized in G1 and S phases of the cell cycle, a clear picture of genome topology in G2/M are still missing. Here we investigated the DNA supercoiling state of the genome in G2/M arrested cells of Saccharomyces cerevisiae using biotinylated 4,5,8-trimethylpsoralen (bTMP) chromatin immunoprecipitation (ChIP) technique. We found that, differently from S-phase cells, Top2 counteracts the accumulation of negative supercoiling in G2/M. The inter and-intra chromosomal interactions of Top2 were revealed with the chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and compared to chromosomal contacts by highthroughput genomic analysis technique (Hi-C). We found that inter-and-intra chromosomal interactions and contacts are mediated by Top2 in S-phase and G2/M in a uniformed manner. The topological context was implicated at the rDNA locus, where changes in DNA supercoils were observed throughout the cell cycle. The rDNA was thus found to be a fundamental genomic region of study, where Top2 interactions were found to be mediated. We propose that Top2 maintains negative supercoils in G2/M and positive supercoils in Sphase. Thus, a different role of Top2 in S-phase and G2/M is suggested in this thesis, where Top2 possibly mediates catenation in S-phase and decatenation in G2/M. A comparison between the genome topological state throughout the cell cycle will therefore be disclosed in this thesis with a main focus on S-phase and G2/M.