Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang - PubMed (original) (raw)

Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang

E Samper et al. EMBO Rep. 2000 Sep.

Abstract

Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals. Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang. In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed. We have measured telomere length in different cell types from wild-type and Ku86-deficient mice. In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang. Interestingly, Ku86-/- cells show telomeric fusions with long telomeres (>81 kb) at the fusion point. These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.

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Figures

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Fig. 1. Chromosome aberrations in Ku86–/– primary MEFs. Cytogenetic alterations detected in Ku86–/– metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3 labelled PNA-telomeric probe. For quantifications see Table I. Blue colour corresponds to chromosome DNA stained with DAPI; yellow and white dots correspond to TTAGGG repeats; red arrows highlight each of the different chromosomal abnormalities shown in the figure.

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Fig. 2. Telomere analysis of littermate wild-type and Ku86–/– MEFs by Q-FISH and FLOW-FISH. (A) Telomere fluorescence of littermate wild-type and Ku86–/– MEFs (passage 1) by Q-FISH and FLOW-FISH (Madrid section in Methods). (B) Telomere length distribution of at least 14 000 telomeres in six different littermate wild-type and Ku86–/– primary MEFs. The histogram depicts similar telomeres in Ku86–/– and wild-type cells. 1 TFU corresponds to 1 kb of TTAGGG repeats.

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Fig. 2. Telomere analysis of littermate wild-type and Ku86–/– MEFs by Q-FISH and FLOW-FISH. (A) Telomere fluorescence of littermate wild-type and Ku86–/– MEFs (passage 1) by Q-FISH and FLOW-FISH (Madrid section in Methods). (B) Telomere length distribution of at least 14 000 telomeres in six different littermate wild-type and Ku86–/– primary MEFs. The histogram depicts similar telomeres in Ku86–/– and wild-type cells. 1 TFU corresponds to 1 kb of TTAGGG repeats.

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Fig. 3. Telomere length analysis of littermate wild-type and Ku86–/– BM and male premeiotic germ cells by Q-FISH. Telomere fluorescence of littermate wild-type and Ku86–/– nuclei by Q-FISH (Leiden section in Methods). Telomere fluorescence of interphase nuclei from two wild-type and Ku86–/– littermates is represented in arbitrary units; each point represents an individual nucleus. Both bone marrow and diploid premeiotic male germ cell nuclei were analysed. BM: wild-type, 28 nuclei; Ku86–/–, 35 nuclei. Testis: wild-type, 27 nuclei; Ku86–/– 39 nuclei.

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Fig. 4. TRF analysis in wild-type and Ku86–/– primary MEFs and BM cells. Three different wild-type (Wt) and Ku86–/– (KO) littermate MEFs (passage 1) were studied, as well as fresh BM samples from two littermate wild-type (Wt) and Ku86–/– (KO) mice. TRF signals were similar in Ku86–/– and wild-type cells. In the case of MEF cultures: I, H and K are three different litters born from heterozygous parents (see also Tables I and II, and Figure 2 for other analyses of the same MEFs). In the case of BM cells, 162, 163, 164 and 165 are mice from the same litter born from heterozygous parents.

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Fig. 5. Normal G-strand overhang in Ku86–/– deficient primary cells. G-strand overhangs in fresh BM cells from two littermate wild-type and Ku86–/– mice are visualized in native gel after hybridization with a (CCCTAA)4 probe (see METHODS). Upon treatment with two different doses of mung bean nuclease (MBN), the G-strand specific signal decreases. (1) and (2) are two different litters. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres.

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Fig. 6. Telomerase activity in wild-type and Ku86–/– MEFs. S-100 extracts were prepared from wild-type (A3 and B1) and Ku86–/– (A1 and C7) MEFs and assayed for telomerase activity. Extracts were pretreated (+) or not (–) with RNase. The protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency.

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