Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA.
Department of Biology and Biotechnology, University of Houston-Clear Lake, Houston, TX, 77058, USA.
G3 (Bethesda). 2021 Dec 8;11(12). doi: 10.1093/g3journal/jkab359.
The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast Saccharomyces cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study, we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, and Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A, and Est2-D671A) suppressed the loss of viability as efficiently as the wild-type Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.
Ku 复合物在真核细胞内执行多种功能，包括保护染色体 DNA 末端免受降解和融合事件的影响、招募端粒酶以及修复双链断裂 (DSB)。在酵母酿酒酵母的细胞中，Ku 复合物基因 YKU70 或 YKU80 的失活会导致表现出端粒缩短和温度敏感生长的突变体。在这项研究中，我们研究了端粒酶过表达抑制 yku 突变体温度敏感性的机制。通过过表达端粒酶的 Est2 逆转录酶和 TLC1 RNA 模板亚基，yku 细胞的存活率得以恢复，但 Est1 或 Est3 蛋白则不行。过表达其他端粒酶和端粒相关蛋白（Cdc13、Stn1、Ten1、Rif1、Rif2、Sir3 和 Sir4）并不能抑制 yku70 细胞的生长缺陷。通过使用几种 TLC1 RNA 缺失衍生物和 Est2 酶突变体评估了抑制的机制特征。三种催化失活的逆转录酶突变体（Est2-D530A、Est2-D670A 和 Est2-D671A）的超生理水平有效地抑制了活力丧失，与野生型 Est2 蛋白一样，而不会诱导细胞衰老。还确定了调节端粒长度的蛋白质的作用。结果支持这样一种模型，即 yku 突变体中的染色体通过涉及保护性端粒帽结构的结构加固的复制独立机制得到稳定。
