Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Division of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Biochim Biophys Acta Mol Cell Res. 2017 Jan;1864(1):191-201. doi: 10.1016/j.bbamcr.2016.11.004. Epub 2016 Nov 9.
Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.
端粒是一种特殊的染色质结构，可防止线性染色体末端的降解和不稳定。虽然端粒酶维持着长串的端粒重复序列，但大多数端粒 DNA 是通过常规的 DNA 复制来复制的。真核生物 DNA 复制的一个基本步骤涉及到起始识别复合物（ORC）与染色质的结合。在人类细胞中，端粒重复结合因子 2（TRF2）被认为在将 ORC 募集到端粒上发挥作用。为了更好地理解 TRF2 介导的 ORC 募集机制，我们在 U2OS 细胞中利用 lacO-LacI 蛋白 tethering 系统，发现异位靶向的 TRF2，但不是 TRF1，可以将 ORC 募集到 lacO 阵列上。我们进一步发现，TRF2 的 TRFH 二聚化结构域，而不是其突变体（二聚化缺陷），足以募集 ORC 和微小染色体维持（MCM）。破坏二聚化的突变也会使全长 TRF2 募集 ORC 的能力受损。使用免疫沉淀和 GST 下拉测定也得到了类似的结果。总之，这些结果表明，二聚化的 TRF2 通过 TRFH 结构域募集 ORC，并刺激端粒处的预复制复合物（pre-RC）形成。
