Erratum

We acknowledge cofunding of the research from the EU ITN project SynCrop (project number 764591), appointment of Luca Mantovanelli

Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast cells exhibit genomic instability, which is also a hallmark of human aging. However, it is unclear how this genomic instability contributes to aging. To shed light on this issue, we investigated endogenous DNA damage in S. cerevisiae during replicative aging and tested for age-dependent sensitivity to exogenous DNA damaging agents. Using live-cell imaging in a microfluidic device, we show that aging yeast cells display an increase in spontaneous Rad52 foci, a marker of endogenous DNA damage. Strikingly, this elevated DNA damage is not accompanied by increased sensitivity of aged yeast cells to genotoxic agents nor by global changes in the proteome or transcriptome that would indicate a specific "DNA damage signature". These results indicate that DNA repair proficiency is not compromised in aged yeast cells, suggesting that yeast replicative aging and age-associated genomic instability is likely not a consequence of an inability to repair DNA damage

The influence of fluorescent protein maturation on FRET measurements in living cells

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet identifying and preventing potential artifacts are needed to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression both in prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster maturing FRET donors. We developed an improved version of the donor mTurquoise2, which matures faster than the parental protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically-encoded FRET sensors

Erratum

We acknowledge cofunding of the research from the EU ITN project SynCrop (project number 764591), appointment of Luca Mantovanelli