Molecular analysis of telomere fusions in Arabidopsis:: multiple pathways for chromosome end-joining
Molecular analysis of telomere fusions in Arabidopsis:: multiple pathways for chromosome end-joining
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DOI：
10.1038/sj.emboj.7600236
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发表时间：
2004-06-02
期刊：
影响因子：
11.4
通讯作者：
Shippen, DE
中科院分区：
文献类型：
--
作者：
Heacock, M;Spangler, E;Shippen, DE
据推测，高等真核生物中严重缩短的端粒的端对端融合是由非同源末端连接（NHEJ）介导的。在这里，我们描述了两种基于 PCR 的方法来监测端粒长度并检查缺乏端粒酶 (TERT) 催化亚基和 DNA 修复蛋白 Ku70 和 Mre11 的拟南芥中功能失调的端粒的命运。引物延伸端粒重复扩增依赖于完整 G 突出端的存在，从而测量功能性端粒长度。检测到的最小功能端粒长度为 300-400 bp。染色体融合连接点的 PCR 扩增和序列分析揭示了融合前功能失调末端的核酸外切消化。在 ku70 tert 突变体中，融合连接处的微同源性发生率比 tert 突变体更高。在三重 ku70 tert mre11 突变体中，仍然检测到染色体融合，但连接处的微同源性不再受青睐。这些数据表明 Ku70 和 Mre11 都有助于高等真核生物中严重缩短的端粒融合。此外，拟南芥利用多种末端连接途径将严重缩短的端粒加工为双链断裂。
End-to-end fusion of critically shortened telomeres in higher eucaryotes is presumed to be mediated by nonhomologous end-joining (NHEJ). Here we describe two PCR-based methods to monitor telomere length and examine the fate of dysfunctional telomeres in Arabidopsis lacking the catalytic subunit of telomerase ( TERT) and the DNA repair proteins Ku70 and Mre11. Primer extension telomere repeat amplification relies on the presence of an intact G-overhang, and thus measures functional telomere length. The minimum functional telomere length detected was 300-400 bp. PCR amplification and sequence analysis of chromosome fusion junctions revealed exonucleolytic digestion of dysfunctional ends prior to fusion. In ku70 tert mutants, there was a greater incidence of microhomology at the fusion junction than in tert mutants. In triple ku70 tert mre11 mutants, chromosome fusions were still detected, but microhomology at the junction was no longer favored. These data indicate that both Ku70 and Mre11 contribute to fusion of critically shortened telomeres in higher eucaryotes. Furthermore, Arabidopsis processes critically shortened telomeres as double-strand breaks, using a variety of end-joining pathways.