Transcription-induced formation of extrachromosomal DNA during yeast ageing

Abstract

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

The adaptive potential of circular DNA accumulation in ageing cells

“…Once replicated, both copies are retained in the mother cell and so the copy number doubles, accounting for the massive abundance of ERCs in aged cells which increase genome size by over 50% (Cruz et al 2018). Surprisingly, we discovered that replication is not required for all circles, as CUP1 circular DNA does not replicate efficiently (despite carrying an annotated replication origin), and is instead generated from chromosomal DNA at such a high rate that simple asymmetric retention of the newly generated copies in the mother cell is sufficient for CUP1 accumulation (Hull et al 2019). However, many circular DNA species do not form at a high rate, nor contain active replication origins and, therefore, the copy number of these species in mother cells will remain static or decrease with age.…”

“…Circular DNA can derive from sites with little or no sequence homology, however, highly repetitive genomic regions, such as the ribosomal DNA (rDNA), telomeres, transposon remnants, and tandemly repeated genes are the largest producers of circular DNA at least in yeast (Moller et al 2015;. Double-strand break repair is the primary source of circular DNA from these repetitive regions although other formation mechanisms have been proposed and may well apply to circular DNA arising from non-repetitive regions (Hull et al 2019;Park et al 1999;Paulsen et al 2018). The existence of circular DNA appears to be common amongst eukaryotes and has so far been reported in fungi, trypanosomes, worms, flies, frogs, mammals and plants (Beverley 1991;Cohen et al 1999;Hotta and Bassel 1965;Koo et al 2018;Shoura et al 2017;Stanfield and Helinski 1976).…”

“…Excitingly we have observed that the rate of copy number variation events including circular DNA formation is not completely random, which may improve this cost-benefit balance for aged cells (Hull et al 2017(Hull et al , 2019. Formation of CUP1 circular DNA, which encodes the copper resistance protein Cup1, is highly dependent on the transcription of the CUP1 locus, which in turn is tightly regulated based on copper in the environment (Hull et al 2019). Therefore, yeast ageing in the presence of copper transcribe CUP1 and so produce many more copies of the CUP1 circular DNA, which pre-adapts these cells to further increases in copper concentration.…”

Extrachromosomal Circular DNA: Current Knowledge and Implications for CNS Aging and Neurodegeneration

“…Another study highlighted the relationship between transcriptional activity and ecDNA formation in the context of aging. In aging budding yeast, the generation of protein-coding eccDNAs was illustrated to be triggered by the transcriptional stimulation of certain genes sensitive to environmental stimuli [115]. Likewise, in yeast cells that aged under copper sulfate treatment, the CUP1 gene was transcribed in a site-specific manner, exclusively leading to the accumulation of CUP1 eccDNA.…”

“…Likewise, in yeast cells that aged under copper sulfate treatment, the CUP1 gene was transcribed in a site-specific manner, exclusively leading to the accumulation of CUP1 eccDNA. Apart from such transcriptional control, the CUP1 eccDNA load was amplified in the aging mother cell by asymmetric segregation and the retention of eccDNAs [115]. Mechanistically, transcription of the CUP1 gene resulted in DSB and, consecutively, eccDNA formation via Sae2, Mre11-and Mus81-dependent DSB-repair mechanisms, suggesting the involvement of recombination events [115].…”