Sannino Vincenzo, Pezzimenti Federica, Bertora Stefania, Costanzo Vincenzo
DNA Metabolism Laboratory, IFOM-The FIRC Institute of Molecular Oncology, Milan, Italy.
DNA Metabolism Laboratory, IFOM-The FIRC Institute of Molecular Oncology, Milan, Italy.
Methods Enzymol. 2017;591:211-232. doi: 10.1016/bs.mie.2017.03.018. Epub 2017 Apr 8.
Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. The possibility of manipulating the protein content by using protein depletion procedures makes egg extract a powerful system to study proteins whose inactivation results in cellular lethality. The egg extract has been increasingly used to study DNA damage response and the coordination of DNA replication with DNA repair. The recent development of advanced imaging techniques based on electron microscopy has allowed the characterization of replication intermediates formed in the absence of essential DNA repair proteins. These studies have been important to understand how cells maintain genome stability under unchallenged and stressful conditions. Here, we present a collection of protocols that have been developed to recapitulate DNA damage response activated by chromosome breakage in egg extract and to isolate replication intermediates for electron microscopy analysis using sperm nuclei or more defined genomic substrates.
尽管在多个系统中已鉴定出许多参与DNA损伤反应和DNA修复的因子，但其生化作用仍知之甚少。非洲爪蟾卵提取物无细胞系统的应用，使得在复杂的生物学背景下对DNA复制和细胞周期事件进行生化剖析成为可能。通过蛋白质去除程序来操纵蛋白质含量的可能性，使卵提取物成为研究那些失活会导致细胞死亡的蛋白质的强大系统。卵提取物越来越多地用于研究DNA损伤反应以及DNA复制与DNA修复的协调。基于电子显微镜的先进成像技术的最新发展，使得对在缺乏必需DNA修复蛋白的情况下形成的复制中间体进行表征成为可能。这些研究对于理解细胞在未受挑战和应激条件下如何维持基因组稳定性非常重要。在这里，我们展示了一系列实验方案，这些方案已被开发出来，用于在卵提取物中重现由染色体断裂激活的DNA损伤反应，并使用精子核或更明确的基因组底物分离复制中间体用于电子显微镜分析。
