INTERPLAY BETWEEN THE DNA HELICASES PIF1 AND RRM3, THE NUCLEASE DNA2 AND THE CHECKPOINT PATHWAYS IN THE MAINTENANCE OF THE DNA REPLICATION FORK INTEGRITY

Eukaryotic cells have evolved the ATR/hCHK1, MEC1/RAD53 kinase-mediated signal transduction pathway, known as replication checkpoint, to protect and stabilize stalled replication forks in human cells and budding yeasts, respectively. rad53 mutants, exposed to high doses of the DNA replication inhibitor hydroxyurea (HU), accumulate hemireplicated, gapped and reversed forks, while treatments with low HU doses induce massive chromosome fragmentation. The aim of my work was to better understand the molecular mechanisms through which Rad53 prevents unusual alterations of the architecture of the stalled replication forks and chromosome fragility, under replication stress. We revealed that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites in unperturbed conditions, are detrimental in rad53 mutants experiencing HU-induced replication stress. Rrm3 and Pif1 ablation synergistically rescues cell lethality, chromosome fragmentation, replisome dissociation, fork reversal and ssDNA gaps formation at the forks of rad53 cells exposed to replication stress. We provide evidence that Pif1 and Rrm3 associate with stalled DNA replication forks and are regulated through Rad53-mediated phosphorylation. Our findings uncover a new replication-stress-induced regulative loop in which Rad53 down regulates the Pif1 DNA helicases at the stalled replication forks. In the second part of this thesis we examined the crosstalk between Rrm3, Pif1, the mediator of the DNA damage checkpoint Rad9 and the nuclease Dna2, during unperturbed DNA replication. The experimental evidence collected in this second part of the project, together with pioneering work previously reported from other laboratories, strongly suggests that Dna2, Pif1 and Rrm3 cooperate to finalize late stages of DNA replication.