| 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 provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.
[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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| Northern Blot of tRNA in Yeast
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Author:
Date:
2013-04-05
[Abstract] tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by ...
[摘要] tRNA是约70-90nt的小RNA。 tRNA与许多其他小RNA不同，因为它们是非常丰富的，这使得难以通过常规Northern印迹研究它们的转录调节。 传统的Northern印迹涉及通过聚合掺入放射性核苷酸，然而，tRNA对于聚合来说太短。 传统的Northern印迹检测RNA水平的变化，然而，tRNA是如此丰富，其水平的小变化将逃脱检测。 由于这些原因，已经使用了放射性尿嘧啶的代谢标记。 然而，代谢标记只能检查总tRNA的变化，但不能区分不同类型的tRNA。 以下方案描述了通过Northern印迹检查单个tRNA基因转录的方法。
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| Synthesis of 5’ end-labeled RNA
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Author:
Date:
2012-07-20
[Abstract] 5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.
[摘要] 5'末端标记的RNA分子是用于分析各种核糖核酸酶的内切和外切核酸活性的有用底物。 这里给出两个方案来合成具有5'PP或5'P部分的P sup 32标记的RNA。 5'外核糖核酸酶通常不在5'PPP RNA上起作用并且需要5'P底物。 某些内切核糖核酸酶如大肠杆菌（大肠杆菌）核糖核酸酶E或枯草芽孢杆菌（枯草芽孢杆菌 ）核糖核酸酶Y可以被5'P部分刺激
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