Steiner B R, Hidaka K, Futcher B
Cold Spring Harbor Labortory, New York 11724, USA.
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2817-21. doi: 10.1073/pnas.93.7.2817.
The est1 mutant was previously identified because it is defective in telomere maintenance and displays a senescent phenotype. To see if Est1 might be a component of yeast telomerase, we examined immunoprecipitated Est1. The yeast telomerase RNA Tlc1 specifically coprecipitated with Est1. Furthermore, the Est1 immunoprecipitates contained a telomerase-like activity. As expected for yeast telomerase, the activity elongated telomeric primers, it required dGTP and dTTP but not dATP or dCTP, and it was sensitive to RNase A. Further evidence suggesting that the activity was telomerase was obtained from experiments using a TLC1-1 mutant strain, which has a mutant telomerase template containing dG residues. The activity immunoprecipitated from TLC1-1 mutant strains incorporated 32P-labeled dCTP, while activity from TLC1 strains did not. Use of different telomeric primer substrates revealed two distinguishable telomerase-like activities: one was dependent on TLC1, and one was not. The TLC1-independent activity may be due to a second yeast telomerase RNA, or it may be some other kind of activity.
est1突变体先前已被鉴定出来，因为它在端粒维持方面存在缺陷，并表现出衰老表型。为了探究Est1是否可能是酵母端粒酶的一个组成部分，我们检测了免疫沉淀的Est1。酵母端粒酶RNA Tlc1能与Est1特异性共沉淀。此外，Est1免疫沉淀产物含有一种类似端粒酶的活性。正如对酵母端粒酶所预期的那样，该活性可延长端粒引物，它需要dGTP和dTTP，但不需要dATP或dCTP，并且对核糖核酸酶A敏感。使用TLC1 - 1突变株进行的实验获得了进一步证据，表明该活性是端粒酶活性，TLC1 - 1突变株具有一个含有dG残基的突变端粒酶模板。从TLC1 - 1突变株免疫沉淀得到的活性可掺入32P标记的dCTP，而从TLC1株得到的活性则不能。使用不同的端粒引物底物揭示了两种可区分的类似端粒酶的活性：一种依赖于TLC1，另一种则不依赖。不依赖TLC1的活性可能归因于酵母的第二种端粒酶RNA，或者它可能是某种其他类型的活性。
