Department of Biochemistry, University of Georgia, Athens, Georgia 30602, USA.
Mol Cell Biol. 2010 Jun;30(12):2971-82. doi: 10.1128/MCB.00240-10. Epub 2010 Apr 19.
Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--TPP1 and the protein that tethers TPP1 to the complex, TIN2--results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.
端粒募集是人类端粒酶功能和调控的关键步骤；然而，募集的分子基础尚不清楚。在这里，我们通过荧光原位杂交（FISH）和染色质免疫沉淀（ChIP）直接研究了端粒酶募集的过程。我们发现，在端粒上发现的庇护复合物的两个成分——TPP1 和将 TPP1 连接到复合物上的蛋白 TIN2 的耗竭，导致端粒酶募集的丧失。另一方面，我们发现，观察到的大多数端粒酶与端粒的结合并不需要 POT1，POT1 是将 TPP1 连接到端粒单链区的庇护蛋白。TPP1 的寡核苷酸/寡糖结合折叠（OB 折叠）缺失会破坏端粒酶的募集。此外，虽然 TPP1 的缺失导致 DNA 损伤因子出现在端粒上，但 DNA 损伤反应本身并不能解释在没有 TPP1 的情况下观察到的端粒酶募集缺陷。我们的研究结果表明，TIN2 锚定的 TPP1 在人类细胞中端粒酶向端粒的募集中起着重要作用，募集不依赖于 POT1 或庇护复合物与端粒单链区的相互作用。
