Investigating protein-rDNA interactions and their link to DNA damage and replicative aging – rDNAproxi

The ribosomal DNA (rDNA) is a highly repetitive genomic region which is essential to initiate ribosome biogenesis. In budding yeast, frequent DNA damages within the rDNA (rDNA damages) triggers the formation of extra-chromosomal rDNA circles (ERCs), which are implicated in replicative aging through a mechanism that is still not clearly understood so far. The objective of this project is to establish which proteins interact specifically with the rDNA sequences during rDNA damages and ERC formation and accumulation. To achieve this goal, we will develop a unique methodology where proteins in close proximity to the rDNA are labelled by biotinylation. Then, we will analyze the biotinylated proteins by mass spectrometry. Hence, we will identify the molecular players involved in rDNA damage repair and in ERC excision and accumulation. This proteomic screen will be complemented by experiments combining fluorescence microscopy and microfluidics. This will allow us to validate the candidates identified by mass spectrometry. In addition, we will monitor, by time-lapse imaging, the dynamics of rDNA damage repair and the accumulation of ERCs in single cells. These results will help to understand: in which cases rDNA damages generate ERCs, why ERCs are toxic to the cell, and how they trigger a loss of nuclear homeostasis during replicative aging. Finally, this work focusing on ERCs in budding yeast will provide a general framework to study the extra-chromosomal DNA circles observed in human cells.