Houghtaling Scott, Tsukuda Toyoko, Osley Mary Ann
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Methods Mol Biol. 2011;745:79-97. doi: 10.1007/978-1-61779-129-1_6.
Multiple types of DNA damage, including bulky adducts, DNA single-strand breaks, and DNA double-strand breaks (DSBs), have deleterious effects on the genomes of eukaryotes. DSBs form normally during a variety of biological processes, such as V-D-J recombination and yeast mating type switching, but unprogrammed DSBs are among the most dangerous types of lesion because if left unrepaired they can lead to loss of genetic material or chromosomal rearrangements. The presence of DSBs leads to a DNA damage response involving activation of cell cycle checkpoints, recruitment of repair proteins, and chromatin remodeling. Because many of the proteins that mediate these processes are evolutionarily conserved, the budding yeast, Saccharomyces cerevisiae, has been used as a model organism to investigate the factors involved in the response to DSBs. Recent research on DSB repair has focused on the barrier that chromatin represents to the repair process. In this article, we describe molecular techniques available to analyze chromatin architecture near a defined DSB in budding yeast. These techniques may be of value to experimentalists who are investigating the role of a novel protein in DSB repair specifically in the context of chromatin.
多种类型的DNA损伤，包括大分子加合物、DNA单链断裂和DNA双链断裂（DSB），对真核生物的基因组具有有害影响。DSB通常在多种生物过程中形成，如V-D-J重组和酵母交配型转换，但未编程的DSB是最危险的损伤类型之一，因为如果不进行修复，它们会导致遗传物质丢失或染色体重排。DSB的存在会引发DNA损伤反应，包括激活细胞周期检查点、招募修复蛋白和染色质重塑。由于介导这些过程的许多蛋白质在进化上是保守的，芽殖酵母酿酒酵母已被用作模式生物来研究参与DSB反应的因素。最近关于DSB修复的研究集中在染色质对修复过程所构成的障碍上。在本文中，我们描述了可用于分析芽殖酵母中特定DSB附近染色质结构的分子技术。这些技术对于正在研究一种新型蛋白质在染色质背景下DSB修复中作用的实验人员可能具有价值。
