| Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
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Author:
Date:
2021-03-20
[Abstract] 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead ...
[摘要] [摘要] 7,8-二氢-8-氧鸟嘌呤（8-oxoG）是由活性氧（ROS）引起的最常见且诱变的氧化DN A损伤之一。由于ROS主要在线粒体的内膜中产生，因此这些细胞器，特别是其中所含的线粒体DNA（mtDNA）受到这种损害的特别影响。消除8-oxoG可能会导致突变，从而导致严重的线粒体功能障碍。为了消除8-oxoG，人体使用了8-氧代鸟嘌呤DNA糖基化酶1（OGG1），它是DNA氧化损伤的主要拮抗剂。但是，先前的研究表明，人类OGG1的活性（h OGG1）随着年龄的增长而减少，导致与年龄相关的8-oxoG积累。更好地了解hOGG1的确切机制可能会导致发现新的靶标，因此对于开发预防性疗法具有重要意义。因此，我们开发了一种实时碱基切除修复测定法，该测定法采用了专门设计的双链报告寡核苷酸来测量分离的线粒体裂解物中hOGG1的活性。这里介绍的该系统与经典测定法不同，在经典测定法中，可以通过实时测量hOGG1活性通过变性丙烯酰胺凝胶进行终点测定。另外，为了确定该双功能酶的每个酶促步骤的活性（N-糖基化酶和AP-裂解酶活性），还可以进行解链曲线分析。使用各种离心步骤从人成纤维细胞中分离线粒体后，将其裂解，然后与专门设计的报告寡核苷酸一起孵育。hOGG1活性的后续测量是在常规实时PCR系统中进行的。
[背景]人体是永久的损害案例。每天约10 ...
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| Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
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Author:
Date:
2018-10-05
[Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ...
[摘要] 核糖核蛋白颗粒（mRNP）是由mRNA和RNA结合蛋白（RBP）组成的复合物，其控制mRNA转录定位，转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件，如poly（A）尾或5'帽结构，这通过应用广泛使用的方法如oligo（dT）纯化来阻止它们的鉴定。在这里，我们描述了基于现有的交联和免疫沉淀（CLIP）方法的修饰的交联亲和纯化方案（cCLAP），以分离mRNP中可被去腺苷酸化，去除和/或部分降解的mRNA，从而开启了检测不同的可能性。非编码RNA（ncRNA）的类型。分离后，将RNA进行衔接子连接，然后进行下一代测序（NGS）。由于快速有效的交联和淬灭步骤，该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体（PB）或应力颗粒（SG）。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀，然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法（Tagwerker et al。，2006; Hafner et al。，2010; Kishore et al。，2011）。 ...
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