title: 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.
authors.affiliation: Department of Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Amita
authors.initials: A
authors.lastname: Vaidya
authors.affiliation: School of Life Sciences and Technology, Tongji University, Yangpu district, Shanghai, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Mao
authors.affiliation: Department of Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Xiao
authors.initials: X
authors.lastname: Tian
authors.affiliation: Department of Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Brianna
authors.initials: B
authors.lastname: Spencer
authors.affiliation: Department of Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Andrei
authors.initials: A
authors.lastname: Seluanov
authors.affiliation: Department of Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Vera
authors.initials: V
authors.lastname: Gorbunova
mesh_terms: D000375:Aging / Q000235:genetics* / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D059766:DNA End-Joining Repair / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005347:Fibroblasts; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D011995:Recombination, Genetic
chemical_list: D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins