Knock-In Reporter Mice Demonstrate that DNA Repair by Non-homologous End Joining Declines with Age

Abstract

Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

Impaired DNA double-strand break repair contributes to the age-associated rise of genomic instability in humans

“…To further examine the fidelity of NHEJ in a more quantitative way, we sequenced the junctions of the rescued plasmids and compared the fidelity between the two groups. Although the process of NHEJ is often associated with deletions or insertions, 4,21,22,23 the chance of acquiring insertions in eyelids fibroblasts is extremely slim (0-15%) (Supplementary Table S2), so it is not feasible to analyze the change of insertion sizes with age in these cell lines. We therefore systematically compared the sizes of deletions at the junction areas.…”

“…3,4,6,11,17,23 Relevant research in humans was mainly focused on age-related change in the recruitment kinetics of essential DNA damage response factors, assayed by immune-staining; 26 age-related change of genomic instability,…”

Do DNA Double-Strand Breaks Drive Aging?

“…In this model, DSBs are generated by the site-specific endonuclease, I-SceI, with the reconstitution of a functional GFP gene a measure of NHEJ activity. NHEJ was shown to significantly decline with age in fibroblasts from heart, lung, kidney, and skin, as well as in astrocytes, with skin showing the most drastic decline (Vaidya et al, 2014). Skin fibroblasts also have the highest rate of senescence in aged mammals (Jeyapalan et al, 2007).…”

Molecular Mechanisms Determining Lifespan in Short- and Long-Lived Species

“…However, DNA repair machinery is not perfect, resulting in accumulation of unrepaired DNA damage, mutations and genomic rearrangements, which in turn may contribute to aging and cancer. In addition, DNA repair efficiency declines with aging and replicative senescence [49–51], which may lead to more rapid accumulation of mutations in aged animals [52, 53]. …”