Profiling DNA end resection and Rad53 kinase activation at a persistent DSB induced in variable sites across yeast genome

In budding yeast, induction of the HO endonuclease by GAL1-10 promoter leads to persistent DSB formation at MAT locus, in the absence of HMR/L donor sequences on chromosome III. Consequently, DNA end resection starts symmetrically from the cut site, leading to robust Rad53 activation. In this condition, cell cycle is delayed in G2/M for several hours, and eventually it is resumed by checkpoint adaptation, a phenomenon accompanied with Rad53 dephosphorylation and inactivation. This genetic system has been employed to identify a sophisticated molecular network for checkpoint inactivation, which involved different DNA repair and checkpoint genes, including also the polo-kinase Cdc5. Although the conservation of mechanisms to mammals and other eukaryotes is poorly studied, adaptation to DNA damage is considered a risky process that can increase chromosome rearrangements and genetic instability, also promoting tumorigenesis. To further study the phenomenon in yeast, we utilized Cas9/gRNA to induce persistent DSB in different positions throughout the genome, focusing on DNA sequence and transcription context. Here we described the production of specific reagents and validate their effectiveness to study checkpoint response, by testing cell viability and Rad53 activation upon Cas9-DSB formation. Along with the analysis of DSB resection and the DNA damage checkpoint response at different DSBs, we also tested by sequencing rare events of repair that can be observed upon corruption of the Cas9/gRNA target sequence. Interestingly, we found that DNA sequence nearby the DSB influences the imprecise repair outcome in the survivors, leading to a variable spectrum of events. We will also show preliminary results regarding the impact of local transcription on DSB processing and the DNA damage response. Our study will provide insights on the molecular response to persistent DSB induced by Cas9/gRNA in yeast, possibly expanding the comprehension of the checkpoint adaptation phenomenon with implication for the study of genome integrity in human and other organisms.