Analysis of Top1 and Top2 contribution to chromosomal DNA replication

Proliferating cells must accurately duplicate their genomes and segregate them into daughter cells in order to preserve their genetic information. Several events can compromise genome integrity during DNA replication, such as dNTPs misincorporation or damaging of the template. DNA replication can also be challenged by the accumulation of topological constraints generated by the separation of the two strands of the DNA double helix. The stress created by the progressing replication machinery can be converted into positive supercoiling (helical overwinding) or precatenates (intertwines the daughter duplexes). Accumulation of supercoiling can cause a block to the progression of replication forks, while accumulation of precatenates represents a physical linkage between chromosomes that impede their correct segregation. DNA topoisomerases are enzymes able to modify the topological state of DNA molecules. Type IB topoisomerases mediate the passage of one strand through another and can efficiently remove supercoiling. Differently, type II topoisomerases mediate the passage of one duplex through another and can act both on supercoiling and precatenates. Moreover, this class of enzymes can unlink sister chromatids prior to chromosome segregation. It is currently unknown what is the cellular response to the accumulation of unresolved topological constraints generated by the replication machinery during S-phase. Cells have evolved different surveillance mechanisms to monitor the correct progression through the different phases of the cell cycle, which are known as checkpoints. In particular, during the S phase, two different checkpoint pathways act to preserve genome integrity: the replication checkpoint and the intra-S phase DNA damage checkpoint. When replication is inhibited or DNA lesions arise in S-phase, a signalling cascade is activated leading to the block of the cell cycle progression and promoting the resolution of replication impediments or the repair of the DNA damage. Rad53 is an essential kinase in this cascade, and is rapidly activated by phosphorylation in response to DNA damage. For this reason the phosphorylation status of this protein can be used as an indicator of checkpoint activation. In this project we investigated the roles of Top1 (Type IB) and Top2 (Type IIA) DNA topoisomerases during the synthesis (S) phase of the cell cycle, and their contribution to genomic DNA replication. By contemporarily inactivating both topoisomerases in budding yeast Saccharomyces cerevisiae cells, we were able to investigate how the accumulation of torsional stress affects replication fork stability and DNA damage checkpoint response.