Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

端粒DNA（Telomeric DNA）是位于染色体末端的短串联重复序列。四膜虫（Paramecium tetraurelia）的端粒DNA由一种易出错的端粒酶（telomerase）合成，该酶带有针对GGGGTT重复序列的RNA模板（RNA template）。我们此前已证实，端粒酶RNA模板核苷酸C52位点处的TTP（胸苷三磷酸，thymidine triphosphate）错掺入，导致野生型端粒中随机分布有30%的GGGTTT重复序列。为更全面地表征四膜虫中可变重复序列的合成过程，我们在体内表达了C52位点突变为A、U及G的端粒酶RNA基因。转化体的从头合成端粒重复序列分析显示，野生型端粒酶中观测到的主要TTP错掺入错误，依赖于模板位点52处的C残基。矛盾的是，其他多种端粒酶RNA模板与对齐区域突变对从头合成端粒的影响，不仅包括保真性的显著改变，还会合成异常的5核苷酸端粒重复序列。突变型四膜虫端粒酶的缺失错误发生与保真性改变，连同野生型酶的错掺入现象，提示端粒酶RNA模板结构域可能类似于多聚核苷酸突变热点——这类热点会导致人类免疫缺陷病毒（human immunodeficiency virus, HIV）的缺乏校对功能的逆转录酶（reverse transcriptase）出现类似错误。