| 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, ...
[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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| Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
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Author:
Date:
2021-03-05
[Abstract] RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein.
The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted ...
[摘要] [摘要] RNA-蛋白质相互作用通常由专门的规范RNA结合域介导。然而，越来越多地观察到通过具有未知特异性的非经典结构域的相互作用，这提出了如何识别RNA靶标的问题。内在的RNA结合特异性的知识有助于理解单个蛋白质的靶标选择性和功能。
所呈现的体外RNA免疫沉淀测定法（vitRIP ）揭示固有RNA使用总细胞RNA池作为分离的蛋白质的结合特异性一个库。从细胞或组织中提取的总RNA与纯化的重组蛋白孵育，免疫沉淀RNA-蛋白复合物，并通过深度测序或定量RT-PCR鉴定结合的转录物。这些RNA中丰富的RNA类和核苷酸频率决定了重组蛋白的固有特异性。该简单而通用的方案可适用于任何细胞类型或组织的其他RNA结合蛋白和总RNA文库。
图形摘要：
图1.体外RNA免疫沉淀（vitRIP ）方案示意图
[背景]真核细胞包含许多不同的RNA类，具有成千上万的RNA种类以及与之相互作用的高度多样化的蛋白质。根据结合的RNA序列或结构的定义以及相互作用中涉及的蛋白质结构域的不同，RNA-蛋白质相互作用可分为特异性和非特异性（Jankowsky和Harris，2015）。越来越多地观察到通过未知特异性的非经典RNA结合结构域进行的RNA相互作用，这提出了如何识别专用RNA靶标的问题。 ...
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| In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
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Author:
Date:
2017-09-20
[Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we ...
[摘要] 真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成，主要通过DNA聚合酶ε和δ以及解旋酶，聚合酶α-引发酶，DNA滑动夹，夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制，以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里，我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that ...
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