Cellular Dynamics Laboratory, CPR, RIKEN, Wako, Japan.
Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan.
Genes Cells. 2021 Jun;26(6):360-380. doi: 10.1111/gtc.12845. Epub 2021 Apr 12.
Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.
小鼠端粒酶和 DNA 聚合酶α-引发酶复合物分别延伸端粒的前导链和滞后链。为了阐明滞后链合成的分子机制，我们研究了 DNA 聚合酶α与小鼠 POT1 端粒结合蛋白（POT1a 和 POT1b）的两个同源物之间的相互作用。酵母双杂交分析和谷胱甘肽 S-转移酶下拉测定表明，POT1a/b 的 C 端区域结合到 p180 的无规卷曲的 N 端区域，p180 是小鼠 DNA 聚合酶α的催化亚基。亚细胞分布分析表明，尽管 POT1a、POT1b 和 TPP1 定位于细胞质中，但与 TIN2 共表达的 POT1a-TPP1 和 POT1b-TPP1 以 TIN2 剂量依赖性方式定位于细胞核中。共免疫沉淀和细胞周期同步化实验表明，POT1b-TPP1-TIN2 与 p180 的结合比 POT1a-TPP1-TIN2 更强，并且该复合物在 S 期积累。荧光原位杂交和邻近连接分析表明，POT1a 和 POT1b 在端粒染色质上与 p180 和 TIN2 相互作用。基于本研究和之前的研究，我们提出了一个模型，即 POT1a/b-TPP1-TIN2 以 TIN2 剂量依赖性方式易位到细胞核中，以靶向端粒，在那里 POT1a/b 与 DNA 聚合酶α相互作用，招募到端粒进行滞后链合成。
