Gómez-González Belén, Ortega Pedro, Aguilera Andrés
Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain.
Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain.
Methods Enzymol. 2021;661:121-138. doi: 10.1016/bs.mie.2021.08.010. Epub 2021 Sep 21.
The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.
DNA双链断裂的修复对于细胞活力和基因组完整性的维持至关重要。当存在完整的姐妹染色单体时，它会被用作首选的修复模板，通过同源重组来恢复遗传信息。尽管由于两条姐妹染色单体难以区分，阻碍了对参与姐妹染色单体重组的因素的研究，但基于DNA重复序列的遗传和分子系统已被开发出来以克服这一问题。特别是，使用能够诱导DNA切口（复制会将其转化为双链断裂）的位点特异性核酸酶，使得通过姐妹染色单体重组对这种复制产生的双链断裂的修复进行特异性研究成为可能。在本章中，我们描述了用于确定这种重组反应的效率和动力学以及重组产物基因定量的详细方案。
