Institute for Cancer Genetics, Department of Genetics and Development and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.
Nucleic Acids Res. 2010 Jan;38(2):467-76. doi: 10.1093/nar/gkp929. Epub 2009 Nov 11.
Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.
端粒的复制需要端粒酶的作用、半保守复制机制以及在穿过端粒 DNA 时复制叉的稳定。尚未通过实验解决脊椎动物端粒是否支持复制起始的问题。使用非洲爪蟾无细胞提取物，我们建立了一个系统来研究线性端粒 DNA 底物中的复制起始。我们显示了 TRF2 与端粒 DNA 的结合，表明外源性 DNA 仅由端粒重复序列组成，可被庇护素成分识别。与端粒结合蛋白的相互作用不足以防止 DNA 损伤反应。值得注意的是，我们观察到前复制复合物蛋白 ORC2、MCM6 和 Cdc6 到端粒 DNA 的调节组装。最重要的是，我们在抑制检查点激活的条件下检测到端粒底物的起始依赖性复制。这些结果表明，前复制复合物在端粒 DNA 内组装，并可转化为功能性起点。
