Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations

Age is the greatest risk factor for developing cells with genome instability and most types of cancer14. There are strong links between aging and genome instability diseases. For example, DNA damage is a driver of aging and the majority of diseases with accelerated or premature aging phenotypes are caused by mutations in DNA repair factors. The overall accumulation of DNA damage is governed by the relative rate of damage formation balanced with the rate of repair, and both are impacted by age. In older cells, DNA damage formation increases and correlates with changes in genome organization and compaction19. At the same time however, the rate of DNA repair decreases. Indeed, cells from older individuals show a lower rate of DSB repair compared to cells from younger individuals32, 51. However, identifying the molecular underpinnings that drive age-related increases in genome instability has remained a challenge.42

Genomic rearrangements are the types of mutations that accumulate with increased age and they arise from defects in DNA double-strand break (DSB) repair.12 The phosphorylation of histone H2AX (γ H2AX) in humans, which corresponds to H2A (γH2A) in budding yeast, is an indirect measure of DSB formation. The level of γH2AX increases with age, yet a unified model for understanding why this occurs is proving to be complex because there are multiple pathways for repairing DSBs. There are two canonical pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), and multiple alternative (alt) pathways. The alt repair pathways are highly mutagenic compared to the canonical pathways and are used when the rates of NHEJ and HR decrease (Figure 1(a)). HR is considered to be error-free as it uses the sister chromatid as a repair template and can faithfully repair a DSB without loss of genetic material. By contrast, NHEJ is error-prone and often incorporates small DNA insertions or deletions at the break site. The relative use of these DSB repair pathways is not uniform across eukaryotes, or uniform in different cell types or developmental stages of the same organism, even under optimal conditions. Yeast cells repair DSBs largely by homologous recombination (HR) whereas human cells rely on NHEJ to a greater extent, however HR and NHEJ are functional in both.

Insights about how DSB repair proceeds during aging have been gained, although working with aging tissue in multicellular organisms remains extremely challenging12, 42. Altered intracellular distribution of Ku70/80 (Ku) and decreased protein levels for Ku and Mre11 have been reported in aging human cells.59, 22, 49, 31 However, changes in protein expression do not necessarily specify which pathway will be used to repair a DSB. Highlighting this case in point, the rate of HR repair decreased in the germline of old flies compared to young flies, yet the expression of HR components increased rather than decreased.7

Cell cycle properties and genome maintenance factors are widely conserved in all eukaryotes and budding yeast has proven to be a robust system for identifying many of the steps in DSB repair. Computational models of aging in unicellular yeast predict that the repair of DNA damage is a critical event impacting cell fitness and replicative lifespan.53, 48 Moreover, recent work has shown a reduction in the levels of HR proteins in older yeast cells as well as a reduction in the use of single-strand annealing (SSA), which is an alternative repair pathway that relies on DNA resection and sequence homology44, 63. However, there is relatively little known about the integrity of DSB repair mechanisms over the replicative lifespan of yeast. Much of the seminal work for monitoring physical events in DSB repair has used the galactose-inducible HO endonuclease system, which creates one site-specific DSB synchronously in the yeast genome.6 Here, we use this well-characterized system to evaluate key steps in DSB repair in different stages of replicative aging. We measured the recruitment of HR and NHEJ repair factors to the HO-induced DSB during aging, determined that DSBs accumulate at the nuclear pore complex in older cells, and measured repair products as replicative age increased by sequencing across the break junction.

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