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, 101 (20), 7624-9

DNA End Joining Becomes Less Efficient and More Error-Prone During Cellular Senescence

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DNA End Joining Becomes Less Efficient and More Error-Prone During Cellular Senescence

Andrei Seluanov et al. Proc Natl Acad Sci U S A.

Abstract

Accumulation of somatic mutations is thought to contribute to the aging process. Genomic instability has been shown to increase during aging, suggesting an aberrant function of DNA double-strand break (DSB) repair. Surprisingly, DSB repair has not been examined with respect to cellular senescence. Therefore, we have studied the ability of young, presenescent, and senescent normal human fibroblasts to repair DSBs in transfected DNA by using a fluorescent reporter substrate. We have found that the efficiency of end joining is reduced up to 4.5 fold in presenescent and senescent cells, relative to young cells. Sequence analysis of end junctions showed that the frequency of precise ligation was higher in young cells, whereas end joining in old cells was associated with extended deletions. These results indicate that end joining becomes inefficient and more error-prone during cellular senescence. Furthermore, the ability to use microhomologies for end joining was compromised in senescent cells, suggesting that young and senescent cells may use different end joining pathways. We hypothesize that inefficient and aberrant end joining is a likely mechanism underlying the age-related genomic instability and higher incidence of cancer in the elderly.

Figures

Fig. 1.
Fig. 1.
Reporter substrate for analysis of NHEJ. (A) The reporter substrate consists of GFP with an artificially engineered intron, interrupted by an adenoviral exon, flanked by restriction sites for induction of DSBs. In this construct, the GFP gene is inactive; however, upon digestion with HindIII or I-SceI enzymes and successful NHEJ, the construct becomes GFP+. (B) Restriction sites used to introduce DSBs. Digestion with HindIII generates compatible cohesive ends. Because I-SceI has a nonpalindromic 18-bp recognition site, cleavage of the two inverted I-SceI sites generates incompatible ends.
Fig. 2.
Fig. 2.
Efficiency of NHEJ declines during cellular senescence. (A) Calibration of the parameters for FACS analysis. Cells were analyzed on a red-versus-green fluorescence plot. The gating for the analysis of green and red cells was set up by using cells transfected with 5 μg of GFP or 5 μg of DsRed vectors, and the cells were transfected with a negative control plasmid expressing the hypoxanthine phosphoribosyltransferase gene (to exclude autofluorescent cells). GFP and DsRed- cells possess autofluorescence and fall along the green/red diagonal, whereas GFP+ and DsRed+ cells appear in separate populations shifted off the diagonal. (B) Relationship between the amount of transfected DNA and the number of GFP+ cells. Cells were transfected with various amounts of circular GFP-Pem1 vector (from 0.01 to 1 μg) mixed with 0.1 μg of pDsRed. After 72 h, the percentages of red and green cells were determined by FACS analysis. The ratio of GFP+ to DsRed+ cells was plotted as a function of the amount of GFP-Pem1 vector. The shaded area indicates the range of GFP+/DsRed+ obtained upon transfection with digested NHEJ substrate. (C)Efficiency of NHEJ in young, presenescent, and senescent WI-38 and HCA2 fibroblasts. Cells were cotransfected with 0.5 μgof HindIII- or I-SceI-digested NHEJ reporter substrate and 0.1 μg of the DsRed expression vector. The numbers of green (GFP+) and red (DsRed+) cells were determined by FACS analysis, and typical FACS traces are shown. The ratio of GFP+ to DsRed+ was used as a measure of NHEJ efficiency. The percent of ligated DNA ends was determined by using the curve shown in B. Hind III, filled bars; I-SceI, open bars. All experiments were repeated at least five times and the SD are shown. Y, young cells; P, presenescent; S, senescent; C, confluent; T, nTERT-immortalized.
Fig. 3.
Fig. 3.
Distribution of deletion sizes among young, presenescent, and senescent cells. A total of 222 clones were analyzed. The clones were grouped according to the deletion sizes. PL, precise ligation. The complete list of deletions in the rescued products is shown in Table 1.
Fig. 4.
Fig. 4.
The frequency of NHEJ events with insertions (A) and without microhomologies (B) among the clones rescued from young, presenescent, and senescent cells. The hatched bar represents the theoretically calculated number of junctions with zero nucleotides of homology if the ends were joined at random. The t test was used to estimate the significance of the differences between the expected frequency of randomly occurring microhomologies and the experimentally observed frequency. *, Statistically significant difference from random.

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