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, 10 (7), e1004511
eCollection

Knock-in Reporter Mice Demonstrate That DNA Repair by Non-Homologous End Joining Declines With Age

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Knock-in Reporter Mice Demonstrate That DNA Repair by Non-Homologous End Joining Declines With Age

Amita Vaidya et al. PLoS Genet.

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of R26NHEJ knock-in mouse model.
(A) The pROSA26PA-NHEJ vector containing NHEJ reporter construct targeted to the ROSA26 genomic sequence. The construct consists of GFP exons separated by the Pem1 intron, interrupted by the killer exon Ad2. Flanking Ad2 are unique I-SceI recognition sites for inducing DSB. Successful NHEJ repair leads to the reconstitution of GFP. Two loxP sites in direct orientation flanking Neomycin/Kanamycin genes (Neo/Kana) and Bacterial origin of Replication (OriC) are located downstream. This vector was targeted by homologous recombination into the endogenous ROSA26 locus of C57BL/6 mouse ES cells. (B) NHEJ construct integrated into ROSA26 locus in the mouse genome. (C) DNA from G418-resistant ES cell clones was digested with BamHI and hybridized to a GFP probe (indicated in B). (D) Founder mice were genotyped using PCR primers indicated in (B). The positive control lanes contain PCR reactions with genomic DNA from ES cell clones shown in (C) as a template. Negative control lane contains PCR reactions with DNA from a wild-type C57BL/6 mouse as template and is followed by a No template control.
Figure 2
Figure 2. NHEJ efficiency declines with age in R26NHEJ mice.
(A) Astrocytes and fibroblasts from heart, kidney, lung, and skin were isolated from 5 young and 5 old mice. After 2 passages, cells were transfected with 5 µg I-SceI for DSB induction and 0.025 µg DsRed to normalize the transfection efficiency. Three days later, cells were analyzed by FACS and NHEJ efficiency was calculated as the ratio of GFP+/DsRed+ cells. For each treatment, 20,000 cells were counted. At least 4 transfections were performed on cells from each mouse and the average NHEJ efficiency from young and old mice was plotted for the individual cell types analyzed. Error bars show s.e.m. (*p<0.05, **p<0.005, t-test). The data for individual mice is shown in Figure S2. (B) Transcription from ROSA26 locus containing the knocked-in NHEJ reporter does not change with age. Total RNA was extracted from the cells of young and old R26NHEJ mice. qRT-PCR was performed using primers homologous to the first exon of GFP and actin primers as the internal control. The experiment was repeated three times and error bars indicate s.d. (C) There is no significant difference in the I-SceI expression between cells from young and old mice. Total proteins were extracted from young and old fibroblasts and astrocytes and the I-SceI levels were analyzed by Western blot with antibodies to the HA-tag. β-actin was used as a loading control. The experiment was repeated three times and a representative image is shown.
Figure 3
Figure 3. Analysis of deletions and insertions at NHEJ junctions in cells from young and old mice.
A total of 300 independent junctions, 30 young and 30 old, for each cell type, were analyzed. The complete list of junction sequences is shown in Table S1. (A) Average deletion size decreases with age in heart fibroblasts and increases in lung and skin fibroblasts. Asterisk indicates significant difference between young and old mice (p<0.05, t-test). (B) Large deletions are more frequently found in old lung and skin fibroblasts and in young heart fibroblasts. The graph shows percentage of NHEJ clones containing deletions larger than 500 bp. Asterisk indicates significant difference between young and old mice (p<0.05, t-test). (C) Average size of insertions increases in astrocytes and decreases in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (p<0.05, t-test). (D) The frequency of insertions decreases with age in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (p<0.05, t-test).
Figure 4
Figure 4. Old heart and lung fibroblasts show increased use of MMEJ.
The complete list of junction sequences is shown in Table S1. (A) The sequenced repair junctions from both young and old cells contain more microhomologies than expected by chance. The hatched bar shows the theoretically calculated frequency of junctions with 1–16 nucleotides of microhomology (16 bp is the longest microhomology in Pem1 intron), if the ends were joined at random. Asterisk indicates that the observed frequency of microhomologies was significantly different from random (p<0.05, t-test). (B) Percentage of NHEJ junctions containing 5–16 bp of microhomology, classified as MMEJ events, increases with age in heart and lung fibroblasts. Asterisk indicates significant difference between junctions from young and old mice (p<0.05, t-test).

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