DNA-PKcs is critical for telomere capping - PubMed (original) (raw)

DNA-PKcs is critical for telomere capping

D Gilley et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

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Figures

Figure 1

Figure 1

DNA-PKcs-deficient MEFs exhibit similar telomere lengths and telomerase activities when compared with wild-type MEFs. (A) Southern analysis of MEFs. Early passage MEFs from independently isolated littermates were prepared from wild-type (lanes 7 and 8), DNA-PKcs+/− (lanes 1, 2, 5, and 6), and DNA-PKcs−/− (lanes 3 and 4). Gel plugs containing genomic DNAs were digested with_Rsa_I and _Hin_fI (odd number lanes) or undigested (even number lanes), fractionated by pulse-field gel electrophoresis, and hybridized with the telomeric specific [TTAGGG]3 probe. The approximate sizes of the products (kb) are indicated based on molecular weight markers. The Southern hybridization signal observed with the [TTAGGG]3 probe under these conditions was sensitive to BAL-31 exonuclease digestion, suggesting that this is telomeric DNA (data not shown). (B) PCR analysis for genotyping. Using the specific primer pairs (see Materials and Methods), wild-type and targeted alleles were amplified as products of 450 bp and 360 bp, respectively. Lanes 2 and 4 show the DNA-PKcs+/− pattern, lane 3 shows the DNA-PKcs−/− pattern, and lane 5 shows the wild-type pattern. Lane 1 contains a size marker. (C) Telomerase activity in DNA-PKcs-deficient MEFs. TRAP assay was performed after 30 PCR cycles on cell extracts (10, 102, and 103 cells) prepared from DNA-PKcs−/− (lanes 1–3), DNA-PKcs+/− (lanes 4–6), and wild-type (lanes 7–9) MEFs. In lanes 10–12, a serial dilution of HeLa cell lysate was run as a positive control for quantitating relative telomerase activity levels. Lane 13 contains a negative control without cell lysate. IC denotes a standard internal control for PCR efficiency.

Figure 2

Figure 2

The frequency distribution of telomere fluorescence in DNA-PKcs−/− cells reveals that telomere length does not vary significantly from wild type and heterozygotes. Data were collected from qFISH studies of metaphase chromosome spreads of the specified genotypes from independently isolated littermates. The x axis depicts the intensity of each signal as expressed in telomere fluorescence units (TFU; 1 TFU = 1 kb of telomeric repeats), and the_y_ axis shows the frequency of telomeres of a given length.

Figure 3

Figure 3

Telomere length does not change in DNA-PKcs−/− kidney cells compared with wild-type cells. (A) Southern analysis using a telomeric-specific probe of kidney cell genomic DNA from independently isolated littermates of each genotype. Gel plugs containing genomic DNA from kidney cells of 6–8-month-old mice were digested with_Rsa_I and _Hin_fI (odd number lanes) or undigested (even number lanes), fractionated by pulse field gel electrophoresis, and hybridized with the telomeric-specific [TTAGGG]3 probe. (B) PCR analysis for genotyping primary kidney cells. The endogenous and targeted alleles were distinguished by using a PCR primer specific for each allele. Lane 1 contains the size marker. Lanes 2 and 3, DNA-PKcs+/+; lane 4, DNA-PKcs+/−; lanes 5 and 6, DNA-PKcs−/−. (C) Telomerase activity is not present in DNA-PKcs+/− and DNA-PKcs−/− kidney cells. Lane 1, no extract as a negative control; lanes 2–4, the activity contained in serial dilutions of HeLa cell lysate; lanes 5–7 DNA-PKcs+/− kidney cell lysate with additions of serial dilutions of HeLa cell lysates; lane 8, DNA-PKcs+/− kidney cell lysate; lanes 9–11, DNA-PKcs−/− kidney cell lysates with serial dilution of HeLa cell lysate; lane 12, DNA-PKcs−/− kidney cell lysate. Numbers indicate approximate cell equivalents used in each assay. IC denotes a standard internal control for PCR efficiency.

Figure 4

Figure 4

FISH analysis of metaphase chromosomes from DNA-PKcs null cells. Representative metaphase chromosome preparations from DNA-PKcs−/− MEFs (A and B) and DNA-PKcs−/− kidney cells (C and D) are shown. rlc, robertsonian fusion configurations; r, ring sister chromatid fusion; t, tri-radial fusion.

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