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Significant correlation of species longevity with DNA double strand break recognition but not with telomere length - PubMed

Antonello Lorenzini  1 ,

Comparative Study

Significant correlation of species longevity with DNA double strand break recognition but not with telomere length

Antonello Lorenzini et al. Mech Ageing Dev. 2009 Nov-Dec.

Abstract

The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSBs) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.

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Figures

Figure 1. Assay for determination of Nuclear DNA-end binding activity

Typical mobility shift assays for the determination of DNA-end binding activity are shown for nuclear protein extracts isolated from human (A), mouse (B) and cow (C) fibroblasts. (D) Quantification of phosphorimager scans of DNA probe binding for each mobility shift. Vertical lines and rectangular callouts represent that amount of protein necessary to bind 50% of the 32P-labeled probe of linear DNA.

Figure 2. Exponential correlation of Nuclear DNA-end binding activity with maximum longevity but not with adult body mass in mammals

Plots of the Log of DNA end binding activity versus species maximum longevity or versus the Log of species maximum longevity are displayed in panels (A) and (B), respectively. Displayed in panels (C) and (D) are plots of the Log of DNA end binding activity versus species body mass or versus the Log of species body mass, respectively. Note that the “Y” axis values are in reverse order because higher binding activity means that less nuclear protein is necessary to bind the same amount of linear DNA probe. Each data point is identified by number, species name, DNA-end binding activity (ug protein to bind 50% of the probe), maximum longevity in years, and average adult body mass in grams are contained in Table 1. Statistical determinations were performed using cultured adult skin fibroblasts (blue diamonds), and thus did not include Chinese hamster CHO lines, human WI 38 fibroblast or human HeLa cells (round red circles). The line in each of the panels represents a regression analysis fit of the data to an exponential function. For the majority of the skin fibroblast cultures the population doublings (PDs) of the culture was 25 and cells had not immortalized. Mouse and Mexican Free Tailed bat (TB) skin fibroblasts had immortalized. TB was used at PDs=40.

Figure 3. Co-variation of longevity and body mass with DNA end binding activity across the available independent comparisons

(a) Phylogeny for the relationships between maximum longevity, DNA end binding and body mass. Branch lengths are not drawn to scale. (b): Direction of variations in maximum longevity, DNA end binding and body mass in the available independent comparisons were determined using the CAIC software package (Purvis et al., 1995). Open, upward-pointed triangles indicate that the two parameters vary in the same direction, while filled, downward-pointing triangles indicate that the parameters vary in opposite directions.

Figure 4. Comparison of Ku 80 and DNA-PKcs protein abundance in human, cow and mouse

Nuclear extracts from mouse, cow, and human were probed for DNA-PKcs, Ku80, serum response factor (SRF) and Histone H3 using antibodies that recognize protein regions that are 100% conserved between these species. The maximum lifespan for these species are 4 years (mouse), 20 years (cow) and 90 years (human). For DNA-PKcs and Ku80 two different film exposures of 5 min (5′) and 5 sec (5′) are shown. The abundance of these proteins related with the capacity to bind DNA ends (Ku80 and DNA-PKcs) reflects the DNA binding capacity of the species. SRF, Histone H3 and the Ponceau Red staining of the membrane (shown only in the region between 37 and 50 kDa) are shown as loading controls.

Figure 5. Comparison of kinetics of DNA double strand break repair in CHO Chinese hamster and WI 38 human fibroblast cells and Western analysis of DNA ligase IV levels

In panel A CHO Chinese hamster (blue circles) and WI-38 human fibroblast cells (red diamonds) growing exponentially in monolayers were irradiated with 20 or 15 Gy of γ-rays, respectively, incubated various times at 37°C in growth medium for repair, and analyzed by asymmetric field inversion gel electrophoresis (Denko et al., 1989; Stamato et al., 1993). Panel B contains a Western blot analysis of the levels of DNA ligase IV in total cellular extracts isolated from hamster (lanes 1 and 2), human fibroblasts (lane 3), LN229 human glioblastoma cells (lane 4), and primary mouse fibroblasts (lane 5). The levels of tubulin are shown as a loading control. Panel C contains a Western blot analysis for DNA ligase IV using nuclear protein extracts from human (lane 1) and hamster cells (lanes 2 and 3). Lane 4 contains extracts from XR1, a negative control hamster CHO-derived cell line that does not express DNA ligase IV (Bryans et al., 1999; Lee et al., 2003).

Figure 6. Telomere length across mammalian species with different longevity

(a) Digested genomic DNA was resolved on a 0.5% agarose gel and probed with an end-labeled (CCCTTA)4 oligonucleotide. Species are ordered by increasing body mass (in grams). DNA marker lengths are shown in kilobases. The first line of the mouse is derived from a wild caught mouse from Pennsylvania, the second line is derived from a wild caught mouse from Idaho. RM = Rhesus monkey. M = DNA ladder. (b) Pulse field gel electrophoresis was used to resolve the telomeres that were too long to be measured in (a), above. Ms = Pennsylvania wild caught mouse, Rabt = rabbit. (c) Fluorescence in situ hybridization (FISH) with a Cy3-conjugated peptide nucleic acid probe (CCCTAA)3 showing that little brown bat telomeres contain internal telomeric repeats. (d) Little brown bat (LBB) telomeres were probed under denaturing conditions (left) or non-denaturing condition (right), only the telomere signals that were detected under non-denaturing conditions were used to estimate mean telomere length. For the majority of the lines the population doublings of the culture were 25 and cells had not immortalized. Mouse and rat cells had spontaneously immortalized.

Figure 7. Correlations of average telomere length to maximum longevity and adult body mass in mammals

(a) Log average telomere length versus Log maximum longevity. (b) Log average telomere length versus Log adult body mass. All the determinations were from cultured adult skin fibroblasts. The species analyzed here are shown in Fig. 6 and 8.

Figure 8. Co-variation of longevity and body mass with telomere length across the available independent comparisons

(a) Phylogeny for the relationships between telomere length, maximum longevity and body mass. Branch lengths are not drawn to scale. (b): Direction of variations in telomere length, maximum longevity and body mass among the available independent comparisons. Open, upward-pointed triangles indicate that the two parameters vary in the same direction, while filled, downward-pointing triangles indicate that the parameters vary in opposite directions.

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