Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast
Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast
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DOI：
10.1074/jbc.m208813200
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发表时间：
2003-01-17
影响因子：
4.8
通讯作者：
Ishikawa, F
中科院分区：
文献类型：
--
作者：
Miyoshi, T;Sadaie, M;Ishikawa, F
Ku 70-Ku 80异二聚体是非同源末端连接途径所必需的保守蛋白质复合物。Ku蛋白也参与端粒的维持，尽管它们的确切作用仍有待阐明。在裂殖酵母中，已经报道了编码Ku 70同源物的基因pku 70（+）。在这里，我们报告的鉴定和表征pku 80（+），基因编码Ku 80。pku 70+（_）和pku 80（+）是有效的非同源末端连接所必需的。我们还发现pku 70和pku 80突变体对甲磺酸甲酯和羟基脲敏感，表明它们在S期的作用。pku 80突变体显示端粒缩短和亚端粒序列的串联扩增，但没有缺陷的端粒位置效应，如先前报道的pku 70突变体。通过使用染色质免疫沉淀分析，我们证明了Pku 70和Pku 80物理相互作用的端粒重复序列和亚端粒序列。有趣的是，这种端粒协会的Pku蛋白是独立的Taz 1，端粒DNA结合蛋白。我们还表明，Pku蛋白不与异位整合的端粒重复序列的内部区域的环状染色体。这些结果表明，DNA的物理末端是必需的定位在端粒的Pku 80。
The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80(+), the gene encoding Ku80. Both pku70+(_) andp ku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku 80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.