Kumanski, S. | Search bioRxiv

bioRxiv2021

A CRISPR-Cas9-based system for the dose-dependent study of DNA double strand breaks sensing and repair

The integrity of DNA is put at risk by different lesions, among which double strand breaks (DSBs) occur at low frequency, yet remain one of the most life-threatening harms. The study of DSB repair requests tools provoking their accumulation, and include the use of chemical genotoxins, ionizing radiations or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have exploited the repetitiveness of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15 or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensing when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.

genetics↗

bioRxiv2021

Negative curvature-promoting lipids instruct nuclear ingression of low autophagic potential vacuoles

Membrane contact sites are functional nodes at which organelles exchange information through moving ions, proteins and lipids, thus driving the reorganization of metabolic pathways and the adaptation to changing cues. The nuclear-vacuole junction of Saccharomyces cerevisiae is among the most extensive and better-known organelle contact sites, described to expand in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a phenomenon that we describe as the most extreme and intimate contact ever reported between nuclei and vacuoles: the vacuole becomes completely internalized in the nucleus. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona-fide drivers of this event. Functionally, we purport that internalized vacuoles are low efficiency ones whose removal from the cytoplasm optimizes cargo interaction with functional vacuoles. Thus, our findings also point to nucleus-vacuole interactions as important for metabolic adaptation. Yet, rather than by inter-organelle exchanges, the underlying mechanism literally concurs with vacuolar sequestration.

cell biology↗