Das-Bradoo Sapna, Bielinsky Anja-Katrin
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Methods Mol Biol. 2009;521:105-20. doi: 10.1007/978-1-60327-815-7_6.
Duplication of eukaryotic chromosomes begins from multiple sites called origins of replication, with DNA synthesis proceeding bidirectionally away from the origin. There is little detailed information available pertaining to whether replication initiates at specific sites or anywhere within a given origin. The development of replication initiation point (RIP) mapping has made it possible to map start sites for DNA synthesis at the nucleotide level. The key step in RIP mapping is the purification of nascent DNA, which is initiated by small RNA primers. For the removal of broken DNA fragments, we utilize lambda-exonuclease, which digests DNA, but leaves nascent strands intact as long as they have the RNA primer still attached. RIP mapping is a sensitive technique and has been successfully applied to single copy loci in both budding and fission yeast, archaebacteria, and human cells. Studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Here, we present a detailed step-by-step protocol for RIP mapping of replication origins in budding yeast.
真核生物染色体的复制始于多个被称为复制起点的位点，DNA合成从起点开始双向进行。关于复制是在特定位点还是在给定起点内的任何位置起始，几乎没有详细信息。复制起始点（RIP）图谱技术的发展使得在核苷酸水平上绘制DNA合成起始位点成为可能。RIP图谱技术的关键步骤是新生DNA的纯化，新生DNA由小RNA引物起始合成。为了去除断裂的DNA片段，我们使用λ核酸外切酶，它能消化DNA，但只要新生链仍附着有RNA引物，就会使其保持完整。RIP图谱技术是一种灵敏的技术，已成功应用于芽殖酵母、裂殖酵母、古细菌和人类细胞中的单拷贝基因座。酵母研究表明，起始因子即起源识别复合体（ORC）的结合位点紧邻复制起始点，这表明ORC将起始机制引导至一个特定位点。在此，我们提供了一份详细的芽殖酵母复制起点RIP图谱技术的分步方案。
