Leman Adam R, Noguchi Eishi
Department of Biology, Duke University, Durham, NC, USA.
Methods Mol Biol. 2014;1170:539-47. doi: 10.1007/978-1-4939-0888-2_30.
A variety of DNA-binding proteins regulate DNA transactions including DNA replication and DNA damage response. To initiate DNA replication in S phase of the cell cycle, numerous replication proteins must be recruited to the replication origin in order to unwind and synthesize DNA. Some replication factors stay at the origin, while replisome components move with the replication fork. When the replisome encounters DNA damage or other issues during DNA replication, the replication fork stalls and accumulates single-stranded DNA that triggers the ATR-dependent replication checkpoint, in order to slow down S phase and arrest the cell cycle at the G2-M transition. It is also possible that replication forks collapse, leading to double-strand breaks that recruit various DNA damage response proteins to activate cell cycle checkpoints and DNA repair pathways. Therefore, defining the localization of DNA transaction factors during the cell cycle should provide important insights into mechanistic understanding of DNA replication and its related processes. In this chapter, we describe a chromatin immunoprecipitation method to locate replisome components at replication origins in human cells.
多种DNA结合蛋白调控包括DNA复制和DNA损伤反应在内的DNA事务。为了在细胞周期的S期启动DNA复制，必须将众多复制蛋白招募到复制起点，以便解开并合成DNA。一些复制因子停留在起点，而复制体组件则随复制叉移动。当复制体在DNA复制过程中遇到DNA损伤或其他问题时，复制叉停滞并积累单链DNA，从而触发ATR依赖的复制检查点，以便减缓S期并使细胞周期在G2-M转换期停滞。复制叉也有可能崩溃，导致双链断裂，从而招募各种DNA损伤反应蛋白来激活细胞周期检查点和DNA修复途径。因此，确定细胞周期中DNA事务因子的定位应该能为深入理解DNA复制及其相关过程的机制提供重要线索。在本章中，我们描述了一种染色质免疫沉淀方法，用于在人类细胞的复制起点定位复制体组件。
