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.
Highlights
The somatic mutation theory of aging posits that the accumulation of unrepaired somatic mutations over time leads to the ‘functional failure’ frequently observed during the course of aging [1]
The non-homologous end joining (NHEJ) reporter cassette [35,36] consists of the GFP gene interrupted by the Pem1 intron and an Ad exon flanked by I-SceI recognition sites (Figure 1A)
We generated the R26NHEJ mouse model, in which a GFP-based NHEJ reporter cassette was knocked-in downstream of the ROSA26 promoter. This is the first instance of a mouse model that can quantify NHEJ repair in multiple mouse tissues
Summary
The somatic mutation theory of aging posits that the accumulation of unrepaired somatic mutations over time leads to the ‘functional failure’ frequently observed during the course of aging [1]. Cellular DNA is a target of various endogenous and environmental insults, leading to DNA damage, of which doublestranded DNA breaks (DSBs) are the most damaging since they can lead to loss of genetic information via deletions or insertions and chromosomal rearrangements via translocations. Accumulation of such genome rearrangements have been observed in aged human and mouse tissues [2,3,4,5,6,7,8]. MMEJ is inherently more mutagenic, leading to deletion of the sequences between microhomology regions [20]
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