Harrell Kailey E, Koury Emily, Smolikove Sarit
Department of Biology, University of Iowa, Iowa City, USA.
Bio Protoc. 2018 Dec 20;8(24). doi: 10.21769/BioProtoc.3130.
DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and cell culture to study the regulation of DSB repair. Up to our studies, this method has not been adapted for use in a whole, live, multicellular organism to study this repair We have recently shown that this system can be used for study of the recruitment of vital repair proteins to microirradiation-induced breaks in the transparent nematode With the integration of microirradiation and imaging technology, we can precisely induce DSBs in target nuclei and study the recruitment of fluorescently tagged repair proteins from the time of damage induction. Whole, live worms are plated and immobilized for targeting of nuclei, and immediately following induction the targeted region can be imaged for up to an hour and a half post-microirradiation. This method is the first that allows for study of DNA repair protein kinetics in an intact organism, which can be adapted in numerous ways to allow for study of repair kinetics in various aspects of the repair process.
DNA双链断裂（DSBs）是毒性损伤，每个细胞都必须准确修复才能存活。DSBs的修复是细胞生命周期不可或缺的一部分，如果修复错误可能导致细胞死亡。激光微照射是一种成熟的技术，已用于酵母、哺乳动物细胞培养以及细胞培养中，以研究DSB修复的调控。在我们的研究之前，该方法尚未适用于完整的、活的多细胞生物体来研究这种修复。我们最近表明，该系统可用于研究重要修复蛋白在透明线虫中微照射诱导的断裂处的募集情况。通过整合微照射和成像技术，我们可以在目标细胞核中精确诱导DSBs，并从损伤诱导时开始研究荧光标记的修复蛋白的募集情况。将完整的活线虫铺板并固定以靶向细胞核，在诱导后紧接着可以对靶向区域进行微照射后长达一个半小时的成像。该方法是第一种能够在完整生物体中研究DNA修复蛋白动力学的方法，它可以通过多种方式进行调整，以便在修复过程的各个方面研究修复动力学。
