Characterization of proteins involved in mitochondrial homologous directed repair in Saccharomyces cerevisiae

Abstract

Our current understanding of the maintenance and repair of mitochondrial DNA (mtDNA) is quite limited, especially when compared to what is known of similar events in the nucleus. It has been shown that double strand breaks (DSBs) are a major source of mutation and deletions in mtDNA, which are associated with increasing predisposition to diseases of great relevance in public health such as cancers, neurodegeneration and normal ageing. Interestingly, the majority of mtDNA deletions are flanked by repeated sequences suggesting that they may result from recombination events. Indeed, previous studies have characterized homologous directed repair (HDR) as one possible mechanism for DSB repair in mitochondria. However, so far, few proteins from the HDR machinery were identified and characterized in mitochondria. In addition, while the DNA damage checkpoint signaling is an important regulator of the HDR pathway in the nucleus, little is known regarding its relevance in mitochondria. Preliminary studies showed that Fun30 and Rad54, previously characterized as importance for the HDR in the nucleus, were enriched in mitochondria fractions from cells treated with the genotoxin MMS. Therefore, the aim of this project is to characterize the importance of the proteins Rad54 and Fun30 and the DNA damage checkpoint signaling for the repair of mtDNA DSBs by using a yeast genetic reporter system to quantitatively measure the occurrence of direct repeat mediated deletions (DRMD) in mtDNA. Moreover, by using a system able to constitutively hyperactivate Rad53, we will investigate the contribution of the checkpoint signaling for mtDNA maintenance.