Gerard Arrey Tané:
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The DNA in eukaryotes is organized inside the nucleus of the cell in discrete and linear molecules, the chromosomes. However, circular DNA molecules originating from the chromosomes lacking centromeres (extrachromosomal circular DNAs or eccDNAs) are increasingly being found in numerous eukaryotic species, showing that genomes are much more malleable than previously thought. While recent descriptive analysis have shown how ubiquitous and heterogeneous these molecules can be, tools and resources to model and study them have lacked. Here, I present two model strategies to study circular DNAs and I use them to investigate their phenotypic effects.
First, I present a method in which we generate endogenous circular DNAs in yeast using the Cre-LoxP system, which allow us to follow circular DNAs during growth of a cell population. In addition, we are able to estimate the reintegration rate back into the linear chromosomes, showing how new alleles formed by circularization can be fixed. Overall, we show how circular DNAs are a source for genetic and phenotypic variability and how their integrations lead to more changes in the chromosomes.
Secondly, I introduce a circular DNA atlas across tissues and ages in mice. While much of the phenotypic effects of circular DNAs have been studied in the context of cancer, much of their effect in somatic healthy tissue has not been yet addressed. In this project, we isolated and sequenced circular DNA from brain, muscle, liver, pancreas and adipose tissue from young (3 month old), adult (12 month old) and old (22 month old) mice. We identify the olfactory receptor genes as being susceptible of recurrent circularization in all tissues, as well as, tissue specific signatures, such as the Ugt1a genes in pancreas.
In this thesis, I also present an in depth literature review, where we describe the factors influencing eccDNA load in mitotically dividing cells. Moreover, I also present a method to increase eccDNA detection during sequencing, where we use CRISPR-Cas9 to eliminate mitochondrial DNA during eccDNA purification.
Lastly, I include a manuscript where we propose an evolutionary impact of eccDNAs in the evolution of mammals. We screened mammalian genomes for changes in synteny explained by formation and reintegration of a circular DNA molecules and showed that up to 6% of mammalian genomes could have originated via this mechanism.