The double strand break (DSB) is a highly toxic form of DNA damage that is thought to be both a driver and consequence of age-related dysfunction. Although DSB repair is essential for a cell’s survival, little is known about how DSB repair mechanisms are affected by cellular age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a repair mechanism for DSBs occurring between direct repeats. Using a single-cell reporter of SSA repair, we measure SSA repair efficiency in young and old cells, and report a 23.4% decline in repair efficiency. This decline is not due to increased usage of non-homologous end joining (NHEJ). Instead, we identify increased G1-phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. We further explore how SSA repair efficiency is affected by sequence heterology and find that heteroduplex rejection remains high in old cells. Our work provides novel quantitative insights into the links between cellular aging and DSB repair efficiency at single-cell resolution in replicatively aging cells.
DNA damage has long been hypothesized to be both a driver and consequence of aging1, 2. Old tissues and cells accumulate DNA damage and mutations3, 4, 5. Such mutations can negatively impact tissue homeostasis and organismal function, and may even increase the risk of cancer6, 7, 8. The DNA double strand break (DSB) represents a major category of DNA damage. Inability to repair a DSB can lead to cell death or genomic instability9. Even when a DSB is repaired, there are mutagenic repair mechanisms that can both increase genetic variability and impact cellular fitness10. Whether cellular aging impairs DSB repair efficiency in single cells remains unknown.
A useful model system to understand how cellular aging affects DSB repair efficiency is that of replicative aging of the budding yeast Saccharomyces cerevisiae11. Replicative lifespan is the number of times a yeast mother cell produces daughters11. This number varies across genetic backgrounds and growth conditions, and is connected to several metabolic pathways12, 13. The connection between DSB repair and replicative lifespan is important for several reasons. To avoid chromosomal instability, most cells that cannot repair a DSB will halt division, and thus have a limited replicative lifespan14. In the other direction, continued cell division after mutagenic DSB repair results in propagation of these mutations to future generations with potentially negative consequences on cell fitness.
Multiple mechanisms exist to repair double strand breaks and their efficiency and ability to function properly could change with replicative age. Among these mechanisms, the non-homologous end joining (NHEJ) pathway involves ligation of the free ends flanking the double strand break15. The other main class of DSB repair mechanisms are the homology-directed repair (HDR) mechanisms, which make use of sequence homology between the break-site and a repair template. Regulation of homology-directed repair can have significant consequences for the genome. This is due to the ability of different HDR mechanisms to change allele copy numbers and lead to recombination between chromosomes. The relative usage and efficiency of different DSB repair mechanisms depend on factors including cell-cycle stage, ploidy, and cell type15, 16, 17, 18. Previous studies have detected changes in repair pathway usage between chronologically old and young tissues, that could indicate age-related changes in repair efficiency19, 20. However, how the repair efficiency of specific DSB repair pathways longitudinally changes in mitotically aging single cells remains unexplored.
Here we assess whether the efficiency of DSB repair via the single-strand annealing (SSA) pathway changes with the age of the host cell. The SSA pathway repairs double strand breaks occurring between direct repeats of an identical sequence, resulting in deletion of the intermediate sequence (Fig. 1a)15. Repetitive sequences play important roles in cellular function, with the rDNA locus being one prominent example21, 22. An age-related change in the efficiency of SSA, which would lead to differences in the copy numbers of the repeated sequence, would be expected to have important consequences for the cell. Since SSA and other homology-directed repair pathways share regulatory aspects such as end-resection and Rad52 recruitment to the repair sites, understanding how SSA efficiency changes with age can provide key insight into whether the efficiency of other homology-directed repair mechanisms would also change with age. Using a singlecell longitudinal approach23 in a haploid genetic background, we measure the efficiency of single-strand annealing repair in young and older cells. We further explore age-related changes in SSA repair efficiency as they relate to age-related changes in cell cycle, non-homologous end joining pathway activity, and in terms of the amount of heterology between the SSA repeats.
Schematics of the experimental system and SSA repair measurements.
a. Diagram showing the steps of single-strand annealing based repair. The direct repeats are highlighted in blue. The upper row shows the situation immediately after the double strand break. b. Schematic showing the setup of the experiment. The yeast replicator device allows replicative aging of yeast to be observed using a microscope. Fresh media is supplied, and waste removed over the course of the movie. A microscope image of a trapped yeast cell is shown above the chip. c. Genetic constructs used to measure SSA. rtTA is expressed from the constitutive PMYO2 promoter. In the presence of doxycycline, rtTA activates expression of I-SceI from a non-leaky PTETO4 promoter. I-SceI cutsite consists of a pair of inverted 18 bp I-SceI sites placed within the SSA reporter. The non-fluorescent 5’ YFP repeat contains only the 5’ 192 bp of YFP. The 3’ YFP repeat is not expressed because it consists of the entire YFP ORF except for the start codon. Between the TEF1 terminator and 3’ YFP repeat is a 2.1 kb stretch of DNA (dotted line), with inverted I-SceI cut sites at a distance of 0.5 kb from the TEF1 terminator. Between the I-SceI cutsite and the 3’ non-functional YFP is an ADH1 promoter driving mCherry (abbreviated by ‘RFP’). Degron-tagged RFP expression below a threshold was used as a reporter for DNA cutting. After SSA repair occurs between the repeats, the resulting product is a full length YFP with a start codon.
