. 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.

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNAdependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs ؊/؊ cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA‐binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X‐ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in ‘tramline’ complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild‐type recombinant GH5. They still bind to H1/H5‐depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA‐binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.

Here we show a correlation between telomere length and organismal sensitivity to ionizing radiation (IR) in mammals. In particular, fifth generation (G5) mouse telomerase RNA (mTR) Ϫ / Ϫ mice, with telomeres 40% shorter than in wild-type mice, are hypersensitive to cumulative doses of gamma rays. 60% of the irradiated G5 mTR Ϫ / Ϫ mice die of acute radiation toxicity in the gastrointestinal tract, lymphoid organs, and kidney. The affected G5 mTR Ϫ / Ϫ mice show higher chromosomal damage and greater apoptosis than similarly irradiated wildtype controls. Furthermore, we show that G5 mTR Ϫ / Ϫ mice show normal frequencies of sister chromatid exchange and normal V(D)J recombination, suggesting that short telomeres do not significantly affect the efficiency of DNA double strand break repair in mammals. The IR-sensitive phenotype of G5 mTR Ϫ / Ϫ mice suggests that telomere function is one of the determinants of radiation sensitivity of whole animals.

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