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Company: Sigma-Aldrich
Catalog#: H8627
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Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
[Abstract]  The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart. [摘要]  DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前,进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同,DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息,包括引发和延长。最后,该测定可用于监测DNA损伤对叉进展,停止和重新启动的影响。

背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰(Prioleau和MacAlpine,2016)控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的,例如DNA损伤,紧密结合的蛋白质复合物和高表达基因的转录(Tourriere和Pasero 2007; Zeman and Cimprich,2013)。真核生物已经制定了不同的策略来应对这种复制压力,包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。

DNA Damage Sensitivity Assays in Caenorhabditis elegans
[Abstract]  C. elegans has served as a genetically tractable multicellular model system to examine DNA damage-induced genotoxic stress which threatens genome integrity. Importantly, the high degree of conservation shared between worms and humans offers the advantage that findings about DNA damage-induced cell cycle arrest/checkpoint response and DNA double-strand break repair in worms are applicable to human studies. Here, we describe simple DNA damage sensitivity assays to quantify the response of C. elegans to diverse types of DNA damaging agents. These assays have provided important insights into the mechanisms of function for factors such as ZTF-8 that are involved in DNA damage repair and response in the C. elegans germline. These DNA damage sensitivity assays rely on ... [摘要]  C。 elegans 作为一种遗传易损的多细胞模型系统,以检查DNA损伤诱导的基因毒性应激,这威胁着基因组的完整性。重要的是,蠕虫和人类之间共享的高程度的保护提供了关于DNA损伤诱导的细胞周期停滞/检查点反应和DNA双链断裂修复蠕虫中的发现适用于人类研究的优势。在这里,我们描述简单的DNA损伤灵敏度测定以量化C的反应。 elegans 到各种类型的DNA损伤剂。这些测定提供了对涉及DNA损伤修复和在C中的应答的因子如ZTF-8的功能的机制的重要见解。 elegans 种系。这些DNA损伤敏感性测定依赖于卵或幼虫致死性的直接读出,并涉及使用各种DNA损伤剂。我们使用产生DNA双链断裂(DSB)的γ-照射(γ-IR),诱导单链断裂的喜树碱(CPT),氮芥(HN <2>),其产生链间交联(ICL),羟基脲(HU),其导致复制叉停止,从而防止DNA合成,和UV-C,其引起光产物(嘧啶二聚体)。参见表1.在例如突变体与野生型相比,在各种DNA损伤剂中观察到的相对灵敏度/抗性之间的比较允许关于潜在修复途径受影响的推论。

DNA Damage Sensitivity Assays with Arabidopsis Seedlings
[Abstract]  We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected. [摘要]  我们描述快速和可重复的灵敏度测定以量化不同基因型的拟南芥幼苗对广泛的DNA损伤剂的反应。 我们应用(1)γ辐射,其产生DNA断裂,(2)bleocin,放射性模拟药物,(3)丝裂霉素C,DNA intrastrand交联剂,(4)羟基脲,DNA合成抑制剂, UV-C,其主要产生光产物。 "真叶试验"和"抗UV试验"基于容易确定的表型作为读数。 使用一组不同的损伤剂结合不同的读数允许相对于参考线(例如野生型)建立相对灵敏度/电阻,确定最有效类型的诱导损伤和潜在的修复途径受影响。