| Charging State Analysis of Transfer RNA from an α-proteobacterium
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Author:
Date:
2020-12-05
[Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces ...
[摘要] [摘要]转移RNA（tRNA）是遗传密码与蛋白质之间的重要纽带。在翻译过程中，tRNA带有其同源氨基酸，并将其传递至核糖体，因此可作为蛋白质合成的底物。为了分析特定tRNA的电荷状态，纯化总RNA并在酸性尿素凝胶上进行分析。然后将分离的RNA转移到膜上并用目标tRNA的探针进行检测。在这里，我们提出了一种改进的协议来分析α-变形杆菌Rhodopseudomonas palustris中的tRNA充电状态 。与传统方法相比，优化了RNA分离步骤以适合这种生物。另外，非放射性平台用于电泳和RNA印迹。这显着减少了此协议所需的时间和精力。
[背景] tRNA的主要功能是，与其他翻译因素的帮助，以确保mRNA的蛋白质的准确的翻译。氨基酰基-tRNA（带电）将氨基酸带到核糖体中以延长肽段，然后释放不带电荷的tRNA。tRNA的充电状态主要取决于可用资源（即氨基酸）及其被核糖体的消耗量。为了分析细胞tRNA的充电状态，已经开发了使用酸性脲凝胶分离总RNA并通过Northern印迹检测感兴趣的tRNA的方法（Janssen等人，2012； ...
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| In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
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Author:
Date:
2017-06-20
[Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase I. This in vitro assay enables us to measure the products for correct vs. oxidized nucleotide insertion, DNA ligation, and ligation failure, i.e., abortive ligation products, as a function of reaction time. This protocol complements our previous publication and describes an efficient way to analyze activities of BER enzymes and the functional interaction between pol β and DNA ligase I in vitro.
[摘要] 我们以前报告说，通过DNA聚合酶β（polβ）的氧化的核苷酸插入可能会在碱基切除修复（BER）（Çağlayan等，2017）期间混淆DNA连接步骤。 在这里，我们描述了在与dGTP或8-氧代-dGTP，polβ和DNA连接酶I相同的反应混合物中研究与DNA连接相结合的polβ核苷酸插入的方法。该体外测定使得我们能够测量产物的正确性 与氧化核苷酸插入，DNA连接和连接失败，即流产结扎产物，作为反应时间的函数。 该协议补充了我们以前的出版物，并描述了一种有效的方法来分析BER酶的活性和polβ和DNA连接酶I在体外的功能相互作用。
【背景】该方案是观察BER路径的最后两个步骤：通过连接酶I通过polβ进行核苷酸插入和DNA连接。使用该方案，在体外在同一反应混合物中测量两种反应作为时间的函数。原始实体是在模拟BER中间体的单核苷酸缺口DNA底物上分析BER酶polβ和DNA连接酶I。这些BER酶与此BER中间体结合并起作用。该方案中使用的DNA底物包括在5'和3'端的荧光标记，使我们能够观察DNA底物的单核苷酸插入和DNA连接。与DNA连接偶联的polβ核苷酸插入模拟了切口DNA中间体从核苷酸插入步骤切换或引导到BER途径期间的连接步骤。使用该方案，也可以在polβ氧化的核苷酸（8-氧代-dGTP）插入后测量连接失败或者终止连接。这通过在BER中间体的5'-末端添加腺苷酸（AMP）基团进行定量来实现。该方案也可用于测量与其他DNA聚合酶和DNA连接酶连接的核苷酸插入。 ...
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| Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells
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Author:
Date:
2015-06-05
[Abstract] We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5´-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make ...
[摘要] 我们先前报道了来自酿酒酵母（Saccharomyces cerevisiae）的细胞提取物中的碱基切除修复（BER）酶DNA聚合酶β（polβ），aprataxin（APTX）和瓣膜内切核酸酶1（FEN1）的酶活性测定 和Wilson，2014）。 在这里，我们描述了一种从脊椎动物细胞中制备细胞提取物以研究这些酶活性用于加工含5'-腺苷酸化糖 - 磷酸酯的BER中间体的方法。 这个新协议补充了我们以前的出版物。 所使用的细胞系是来自具有Oclomotor Apraxia 1型（AOA1）疾病患者，野生型和APTX无效DT40鸡B细胞和小鼠胚胎成纤维细胞（MEF）细胞的共济失调的野生型和APTX缺陷型人淋巴母细胞。 这个协议是一个快速和有效的方式来制作脊椎动物细胞提取没有使用商业套件。
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