A genome‐wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

Abstract To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.

aged 1 L culture was harvested. The aged cells were processed similarly to the young cells, with slight modifications because of the higher number of cells due to the presence of daughter cells. For beading, the cells were split into 4 different 5 ml LoBind tubes, and 50 µl streptavidin coated BioMag beads were added to each tube. For magnetic sorting, two glass tubes were used and cells were washed four times.
For both young and aged samples, colonies were counted after 2 d of growth at 30°C and the spontaneous forward mutation frequencies at the CAN1 locus were determined.
Expected mutation frequencies in aged cells were calculated as previously described (Patterson & Maxwell, 2014).

Bud scar detection and counting
Purified mother cells (see above) were stained with propidium iodide (PI) (Sigma) to identify viable cells and with Calcofluor White (Fluorescent Brightener 28, Sigma) to detect bud scars. 100 µl of purified mother cells in PBS (~5 x 10 5 cells) were stained with 2 µl of a 2 mM PI (Sigma) solution for 30 min at 30°C. Cells were then washed with ddH2O, fixed in 500 µl of 3.7% formaldehyde for 30 min at room temperature, washed with PBS, resuspended in 100 µl PBS and stored at 4°C. Just before imaging, cells were stained with Calcofluor White for 5 min at room temperature, washed with PBS and resuspended in 5-10 µl PBS. Images were acquired using a DeltaVision Elite imaging system (Applied Precision (GE), Issaquah, WA, USA) composed of an inverted microscope (IX-71; Olympus) equipped with a Plan Apo 100X oil immersion objective with 1.4 NA, InsightSSITM Solid State Illumination, excitation and emission filters for DAPI and A594, ultimate focus and a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ, USA). Stacks of 30 images with 0.2 µm spacing were taken at an exposure time of 5 ms at 10% intensity for DAPI (Calcofluor White staining) and 50 ms at 32% intensity for A594 (PI staining). Reference bright-field images were also taken. Fluorescent images were subjected to 3D deconvolution using SoftWoRx 5.5 software (Applied Precision). Processing of all images was performed using Fiji (ImageJ, National Institute of Health) (Schneider et al., 2012). Bud scars from at least 50 PI-negative cells (which were alive after magnetic sorting) were manually counted for each sample to determine the cells' replicative age.

DNA damage sensitivity of young and aged cells
DNA damage sensitivity of young and aged wt and pex19∆ cells was assessed as described in (Novarina et al., 2017). Exponentially growing cultures were diluted to a concentration of 4 × 10 4 cells/ml and estradiol was added to a final concentration of 1 µM to induce the MEP. Half of the culture was treated 2 h after induction by estradiol (young cells), while the other half was incubated for 20 h shaking at 30 °C (aged cells). Young and aged cells were mock-treated or treated with H2O2 or MMS at the indicated doses. Cells were diluted tenfold prior to plating on four plates (technical replicates) per dose/time point. Plating volumes were adjusted for young and aged cells to obtain ∼100 colony forming units per plate. Mock-treated aged cells were also plated on control plates containing 1 µM estradiol to detect escapers. If the escaper frequency in the culture was higher than 10%, the experiment was discarded.  Figure S1. Exclusion of strains that escaped before the beginning of the screen. When serial dilutions of strains from the MEP-YKO collection are spotted in the presence of estradiol, growth of MEP-proficient strains is restricted, while escaper strains grow normally. An example of one MEP-proficient strain and one escaper is shown.   Figure S3. ICE2, ATG23, and ROX3 did not validate as age-specific mutation suppression genes.  (B) Age-dependent mutation frequencies at the CAN1 locus in wt (replicative age ~17) and pex3∆ (replicative age ~13) cells. The difference between observed and expected mutation frequency is plotted. For the wt, the mean value from four independent experiments is plotted. Error bars represent standard error. For pex3∆, only one experiment is shown. Figure S5. Aged pex19∆ cells do not display increased sensitivity to H 2 O 2 or MMS. Survival curves for young (t = 2 h) and aged (t = 20 h, corresponding to age ~13) wt and pex19∆ MEP cells exposed to H 2 O 2 (A) or MMS (B) at the indicated doses. The duration of the H2O2 and MMS treatments were 30 min and 20 min, respectively. Mean values from at least three independent experiments are plotted. Error bars represent standard error.   ICE2 integral ER membrane protein 3,3 x 10 -7 3,5 x 10 -7 2,6 x 10 -1 NO PEX19 biogenesis of peroxisomes 2,9 x 10 -7 3,4 x 10 -7 1,2 x 10 -1 YES ATG23 autophagy and cytoplasm-to-vacuole targeting 3,5 x 10 -7 3,2 x 10 -7 7,1 x 10 -3 NO ROX3 RNA polymerase II subunit 3,5 x 10 -7 2,7 x 10 -7 4,9 x 10 -3 NO wild type 2,6 x 10 -7 2,9 x 10 -7 7,7 x 10 -4  Table S3. Escaper formation is not always caused by mutations at the cre-EBD78 locus.