Gadaleta Mariana C, Iwasaki Osamu, Noguchi Chiaki, Noma Ken-Ichi, Noguchi Eishi
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N 15th Street, Philadelphia, PA, 19102, USA.
Methods Mol Biol. 2015;1300:169-86. doi: 10.1007/978-1-4939-2596-4_12.
DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors.
DNA复制与DNA修复过程紧密耦合，以维持基因组完整性。在DNA复制过程中，复制叉会遇到各种障碍，包括DNA损伤/加合物、二级结构和程序性叉阻断位点，这些都难以复制。复制叉还会与转录机器发生碰撞，转录机器与复制体复合物共享模板DNA。在这些条件下，复制叉停滞，导致复制压力和/或叉崩溃，最终导致基因组不稳定。克服这些复制问题的机制仍然难以捉摸。因此，研究DNA修复和复制因子如何在难以复制的染色体区域被招募和协调是很重要的。在本章中，我们描述了一种染色质免疫沉淀方法，用于定位裂殖酵母粟酒裂殖酵母DNA复制过程中DNA修复所需的蛋白质。该方法也可以很容易地用于研究复制体成分或染色质相关因子。
