Quantitative Insights into Age-Associated DNA-Repair Inefficiency in Single Cells

Abstract

Although double-strand break (DSB) repair is essential for a cell's survival, little is known about how DSB repair mechanisms are affected by age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a DSB repair mechanism. We measure SSA repair efficiency in young and old yeast cells and report a 23.4% decline in repair efficiency. This decline is not due to increased use of non-homologous end joining. Instead, we identify increased G1 phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. Expression of 3xCLN2 leads to higher SSA repair efficiency in old cells compared with expression of 1xCLN2, confirming the involvement of cell-cycle regulation in age-associated repair inefficiency. Examining how SSA repair efficiency is affected by sequence heterology, we find that heteroduplex rejection remains high in old cells. Our work provides insights into the links between single-cell aging and DSB repair efficiency.

Keywords: DNA repair; aging; double-strand break; microfluidics; microscopy; replicative lifespan; single cell; single-strand annealing; systems biology; yeast.

Figure 1.. 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 orange. 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 is 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 P_MYO2 promoter. In the presence of doxycycline, rtTA activates expression of I-SceI from a non-leaky P_TETO4 promoter. The I-SceI cut site consists of a pair of inverted 18 bp I-SceI sites (black arrows) placed within the SSA reporter (rc, reverse complemented). 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 cut site and the 3′ non-functional YFP is an ADH1 promoter driving mCherry (abbreviated as “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.

Figure 2.. Measuring SSA Repair Efficiency under Perfect Sequence Homology

(A) Error bars correspond to mean ± SEM of the average age within replicates. Red points represent the age of each cell at the beginning of the doxycycline treatment. The mean age of old cells (n = 2 replicates) was 18.1 generations, while the mean age of the young cells (n = 2 replicates) was 2.6 generations. (B) YFP trajectories are shown for two young sample cells, with the cell with the red trajectory representing successful SSA repair. (C) The fraction of cells with successful SSA repair was calculated for each experimental replicate. Shown are mean ± SEM (n = 2) by age group for the fraction of repaired cells. The old cell replicates had a mean repair efficiency of 71.8% compared with a mean repair efficiency of 93.8% for the young cell replicates. The p value of 0.003 is for a two-sided Fisher’s exact test for association between repair and age on pooled data. (D) Shown are the mean ± SEM (n = 2) of the replicate-based averages of single-cell average budding intervals for time windows before and after doxycycline addition. Pre-dox corresponds to the 4 h time window before doxycycline addition. Post-dox corresponds to the 9 h time window after doxycycline addition. At both young and old ages, the strain carrying the SSA repair reporter exhibits increased budding interval relative to the control strain, indicative of DNA damage-induced cell-cycle arrest (from 74 ± 1 min to 102 ± 8 min in young cells with the cut site; from 84 ± 9 min to 153 ± 23 min in old cells with the cut site).

Figure 3.. Directly Measuring I-SceI Cutting Efficiency Using a Degron-Tagged mCherry (“RFP”) in the SSA Repair Cassette

(A) An RFP cutoff was applied to cells from two strains containing a degron-tagged mCherry (abbreviated “RFP”) within an SSA repair reporter: one strain contained an I-SceI cut site while the other lacked the cut site. RPF measurements from one such cut-site-positive cell are shown along with the RFP cutoff (red line). For each cell analyzed, the duration of RFP absence was quantified between the first occurrence of below-cutoff RFP level and the end of the 9 h post-doxycycline time window. (B) The duration of RFP absence is shown for old and young cells of the strain containing a degron-tagged RFP within an SSA repair reporter and old cells of the same strain missing the I-SceI cut site. Forty-one of the 58 old cells (71%) analyzed from the second strain missing the cut site had a duration of RFP absence quantified as 0 min, compared with 1 of the 81 old cells from the first strain carrying the cut site. Using pooled data from two independent experiments, the boxplots show the distribution of the single-cell RFP⁻ durations for each cell. (C) For each cell that was missing RFP (expression below the cutoff) at the 9 h time point after doxycycline addition, all RFP values prior to the drop below the cutoff were averaged for that cell. Red points correspond to these single-cell averages. Using pooled data from two independent experiments, the boxplots show the distribution of the single cell-averaged RFP values corresponding to each cell’s RFP⁺ period. The RFP values corresponding to the RFP⁺ period are similar between old cells of the two degron-tagged SSA reporter strains, with and without the cut site.

Figure 4.. The Age-Associated Decline in SSA Repair Efficiency Is Due to Changes in Cell-Cycle Progression in Old Cells

(A) dnl4Δ cells fail to show an increase in SSA repair efficiency when old. The mean repair efficiency for old cells (n = 2 replicates) was 65.0%, while for young replicates (n = 2), it was 90.4%. The error bars show the mean ± SEM (n = 2) of these replicate-based statistics. The p value of 0.0016 is for a two-sided Fisher’s exact test to test for association between repair and age on pooled data. (B) The dnl4Δ strain shows a large increase in budding interval after doxycycline treatment regardless of age (from 71 ± 1 minto 103 ± 16 min in young cells; from 77 ± 1 min to 156 ± 16 min in old cells). Shown are the mean ± SEM (n = 2) of replicate-based averages of single-cell average budding intervals during the 4 h pre-doxycycline addition and 9 h post-doxycycline addition time window. (C) The strain containing a degron-tagged mCherry (“RFP”) within the SSA repair reporter has an SSA repair efficiency of 84.0% for old cells (n = 3 replicates) and 89.2% for young cells (N = 2 replicates). Shown are mean ± SEM of replicate-based repair efficiencies. The p value of 0.58 is for a two-sided Fisher’s exact test to test for association between repair and age on the pooled data. (D) Average budding intervals during the 9 h post-doxycycline time window are similar in old cells of the SSAcontrol strain (no cut site) carrying degron-tagged mCherry (“SSAcontrol+RFPdegron”) and old cells of the SSAcontrol strain carrying stable mCherry (“SSAcontrol+stableRFP”). Shown are mean ± SEM (n = 2) of replicate-based averages of single-cell average budding intervals, pre-dox (during the 4 h time window before doxycycline addition) and post-dox (during the 9 h time window after doxycycline addition). In old cells, the average post-dox budding interval was 115 ± 6 min for the RFPdegron-carrying control strain and 111 ± 8 min for the stable RFP-carrying control strain. (E) Average budding intervals occurring after generation 15, measured for old cells of the SSA control strains (no cut site), with stable RFP or degron-tagged RFP. Only cells of age 15 generations or older at the time of doxycycline addition were used. Mean ± SEM (n = 2) of replicate-based averages of single cell-averaged budding intervals are shown. The mean values are 117 ± 1 min for the strain with the stable RFP and 96 ± 6 min for the strain with the degron-tagged RFP. (F) SSA repair efficiency is increased in old cells of the strain carrying three copies of the CLN2 gene. Shown are the mean ± SEM (n = 2) by age group for the fraction of repaired cells. The old cell replicates had a mean repair efficiency of 88.7% (n = 2). The young cell replicates had a mean repair efficiency of 83.8% (n = 2). The p value of 0.54 is for a two-sided Fisher’s exact test to test for association between repair and age on pooled data.

Figure 5.. Measuring SSA Repair Efficiency under 3% Sequence Heterology

(A) Old cells show a decline in SSA repair efficiency relative to young cells. Repaired cell fraction was measured for each replicate to quantify SSA repair efficiency. The mean and SEM (n = 2 or 5) of these replicate-based statistics are shown. The replicates (n = 5) of the old cells had a mean repair efficiency of 27.5%, while the replicates (n = 2) of the young cells had a mean repair efficiency of 34.9%. The p value of 0.28 is for a two-sided Fisher’s exact test to test for association between repair and age on pooled data. (B) Shown are the mean ± SEM (n = 2 or 5) of replicate-based averages of single-cell average budding intervals for the 4 h time window before and the 9 h time window after doxycycline addition. Cells exhibit an especially large increase in cell-cycle duration after doxycycline treatment (from 75 ± 8 min to 161 ± 18 min for young cells; from 74 ± 3 min to 169 ± 7 min for old cells).