Fig 1.
Telomere sequence divergence events in pre-senescent est2Δ cells are independent of Rad52.
Telomere VI-R was amplified and sequenced from clonal populations of est2Δ (derived the sporulation of CCY16 and CCY8), est2Δ rad52Δ (derived the sporulation of CCY16), est2Δ rad59Δ (derived the sporulation of CCY8), and est2Δ pol32Δ cells (derived from the sporulation of CCY18), ~30 population doublings after isolation of haploid spores. Each bar represents an individual telomere and bars are sorted by the length of the undiverged sequence (black portion of each bar). The light gray portion of each bar represents the diverged region.
Fig 2.
Deletion of RAD52 does not increase telomere truncation events.
Telomere VI-R was amplified and sequenced from clonal populations of wild-type and rad52Δ cells (derived from the sporulation of W8758), ~35 population doublings after the isolation of haploid spores. Each bar represents an individual telomere and bars are sorted by the length of the undiverged sequence (black portion of each bar). The light gray portion of each bar represents the diverged region. The red line highlights 125 nt. The number of telomeres with less than 125 nt of undiverged sequence is shown.
Fig 3.
BIR does not fully account for the role of Rad52 in preventing accelerated senescence.
Senescence rates were measured by serial passaging est2Δ, est2Δ pol32Δ, and est2Δ rad52Δ strains (derived from the sporulation of CCY155) in liquid culture. Cell density was measured each day after 24 h of growth in liquid culture, followed by dilution to 2 x 105 cells/ml. Mean ± SE for five independent spore isolates for est2Δ and ten isolates for both est2Δ pol32Δ and est2Δ rad52Δ is shown.
Fig 4.
The strand annealing activity of Rad52 is important for preventing accelerated senescence and for the formation of type II survivors.
(A–C) Senescence rates were measured in liquid culture by serial passaging of haploid meiotic progeny of the indicated genotypes, derived from the sporulation of CCY114 (A), CCY126 (B), or CCY127 (C). Cell density was measured each day after 24 h of growth in liquid culture, followed by dilution to 2 x 105 cells/ml. Mean ± SE for each genotype is shown. At least eleven independent isolates for each genotype were followed in A, and at least four in B and C. (D) Telomere Southern blot of est2Δ and est2Δ rad52-R70A survivors obtained from the liquid culture senescence assay from A. est2Δ rad52-R70A survivors were analyzed on average 13.9 PDs after the point of maximum senescence. Type I survivors exhibit short telomeres and strong hybridization at 5.2 kb and 6.7 kb, which is due to amplification of the tandemly repeated Y′ short and Y′ long elements, respectively. The telomeres of type II survivor are extended and very heterogeneous in size.
Fig 5.
Hyper- and hypoacetylation of H3K56 causes accelerated senescence.
(A, B) Senescence rates were measured in liquid culture by serial passaging of haploid meiotic progeny of the indicated genotypes, derived from the sporulation of CCY136 (A) and CCY143 (B). Mean ± SE for at least five independent spore isolates for each genotype is shown. (C, D) Telomere Southern blot of survivors of the indicated genotypes obtained from the liquid culture senescence assays from A and B. The parental diploids (CCY136 and CCY143) were included in the blots. est2Δ hst3Δ hst4Δ and est2Δ rtt109Δ survivors were analyzed on average 21.5 and 19.5 PDs, respectively, after the point of maximum senescence. Type I survivors exhibit short telomeres and strong hybridization at 5.2 kb and 6.7 kb, which is due to amplification of the tandemly repeated Y′ short and Y′ long elements, respectively. The telomeres of type II survivor are extended and very heterogeneous in size.
Fig 6.
A model to describe the known Rad52-mediated mechanisms that function at telomeres during replicative senescence.
Replication forks traveling through subtelomeric and telomeric sequences frequently encounter impediments to their progression. A fork collapse would leave a truncated telomere, which can be elongated by telomerase (far left). In the absence of telomerase (gray box), the truncated telomere can be repaired by BIR. Fork impediments can be dealt with via error-free PRR. Blocks of lagging and leading strand synthesis can be repaired via Rad5-independent and Rad5-dependent error-free PRR, respectively.
Table 1.
List of S. cerevisiae strains used in this study.
All strains are ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5 unless indicated otherwise.