Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
J Mol Biol. 2011 Jul 1;410(1):10-7. doi: 10.1016/j.jmb.2011.04.049. Epub 2011 May 9.
Telomeres are nucleoprotein complexes that cap and protect the ends of linear chromosomes. In humans, telomeres end in 50-300 nt of G-rich single-stranded DNA (ssDNA) overhangs. Protection of telomeres 1 (POT1) binds with nanomolar affinity to the ssDNA overhangs and forms a dimer with another telomere-end binding protein called TPP1. Whereas most previous studies utilized telomeric oligonucleotides comprising single POT1-TPP1 binding sites, here, we examined 72- to 144-nt tracts of telomeric DNA containing 6-12 POT1-TPP1 binding sites. Using electrophoretic mobility gel shift assays, size-exclusion chromatography, and electron microscopy, we analyzed telomeric nucleoprotein complexes containing POT1 alone, POT1-TPP1, and a truncated version of POT1 (POT1-N) that maintains its DNA-binding domain. The results revealed that POT1-N and POT1-TPP1 can completely coat long telomeric ssDNA substrates. Furthermore, we show that ssDNA coated with human POT1-TPP1 heterodimers forms compact, potentially ordered structures.
端粒是一种核蛋白复合物，能够覆盖并保护线性染色体的末端。在人类中，端粒的末端是 50-300 个核苷酸的富含 G 的单链 DNA（ssDNA）突出端。端粒保护蛋白 1（POT1）以纳摩尔亲和力结合到 ssDNA 突出端，并与另一种称为 TPP1 的端粒末端结合蛋白形成二聚体。虽然之前的大多数研究都利用包含单个 POT1-TPP1 结合位点的短寡核苷酸来研究端粒，但在这里，我们研究了含有 6-12 个 POT1-TPP1 结合位点的 72-144 个核苷酸的端粒 DNA 片段。我们使用电泳凝胶迁移率变动分析、尺寸排阻层析和电子显微镜分析了仅含 POT1、POT1-TPP1 和保持其 DNA 结合结构域的 POT1 截短蛋白（POT1-N）的端粒核蛋白复合物。结果表明，POT1-N 和 POT1-TPP1 可以完全覆盖长的端粒 ssDNA 底物。此外，我们还表明，用人类 POT1-TPP1 异二聚体覆盖的 ssDNA 形成了紧凑的、可能有序的结构。
