An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila.
An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila.
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DOI：
10.1091/mbc.e11-11-0952
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发表时间：
2012-06
影响因子：
3.3
通讯作者：
Yao MC
中科院分区：
文献类型：
--
作者：
Lin IT;Chao JL;Yao MC
在四膜虫中，DNA重排以接合方式发生，并导致大的特定DNA片段的缺失和染色体的碎裂。KU80同源物是NHEJ途径的一个组成部分，对于这两个过程以及在核分化过程中形成专门的DNA消除异染色质结构是必不可少的。程序性DNA重排是存在于许多生物体中的重要过程。在纤毛原生动物嗜热四膜虫中，DNA重排发生在有性交配过程中，导致数千个特定DNA片段的缺失和染色体的碎裂。在本研究中，我们发现Ku80同源物是DNA修复的非同源末端连接过程中的一个保守成分，对这两个过程是必不可少的。在接合过程中，TKU80高度表达并定位于新的大核，在那里发生DNA重排。同核体TKU80-基因敲除突变体不能完成接合并产生后代，并在两微核/两大核阶段受阻。对他们的DNA分析显示，他们未能完成DNA缺失。然而，DNA切割步骤似乎已经发生，环状切除的DNA的存在证明了这一点。此外，染色体断裂或从头端粒增加也受到影响。突变体似乎积累了末端脱氧核苷酸转移酶dUTP缺口末端标记检测到的游离DNA末端，导致发育中的大核中的大多数DNA降解。这些发现表明，Tku80p可能在DNA切割发生后起到末端保护作用。出乎意料的是，通常与DNA重排相关的大型异染色质结构在没有TKU80的情况下未能形成。综上所述，这些结果提示了Tku80p的多种作用，并表明Ku依赖的DNA修复途径参与了四膜虫的程序性DNA重排。
In Tetrahymena, DNA rearrangements occur in conjugation and lead to the deletion of large specific DNA segments and fragmentations of the chromosomes. The Ku80 homologue, a component of the NHEJ pathway, is essential for these two processes and for forming the specialized DNA elimination heterochromatin structures during nuclear differentiation. Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.