| Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
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Author:
Date:
2021-03-05
[Abstract] In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to ...
[摘要] [摘要]在细菌中，停滞的DNA复制叉的重新启动需要DN A解旋酶PriA 。PriA可以识别并重塑废弃的DNA复制叉，在3'到5'方向展开DNA，并促进解旋酶DnaB加载到DNA上以重新开始复制。ssDNA结合蛋白（SSB）通常存在于废弃的叉子上，从而保护ssDNA免受核酸酶的破坏。该研究是基于所述测定法离解结，表面等离振子共振，单分子FRET，和x射线晶体结构已经揭示的解旋酶活性PRIA ，SSB- PriA相互作用以及PriA解旋酶的结构信息。在这里，我们使用原子力显微镜（AFM）可视化了在不存在ATP的情况下在有或没有SSB的情况下PriA和带有或不带有SSB的DNA底物之间的相互作用，以描绘PriA在其ATP催化的DNA解链反应之前的底物识别模式。该协议描述了获取高分辨率AFM图像的步骤以及数据分析和表示的细节。
[背景]当DNA复制遇到障碍或断裂时，需要对其进行修复并随后重新启动（Kogoma，1997； Cox等，2000； McGlynn和Lloyd，2002；G abbai和Marians，2010； Michel等，2018）。 ）。在细菌中，DNA解旋酶PRIA通过识别废弃DNA复制叉，从而便于重新组装的介导这一过程复制体的解旋酶和装载DNAB （Wickner和赫维茨，1975; Zavitz和Marians，1992; ...
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| EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
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Author:
Date:
2021-02-20
[Abstract] Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer’s. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be ...
[摘要] [摘要]转录错误可通过改变表观基因组并引起mRNA的错误整合而严重影响生物体内的代谢过程，从而将其翻译为异常的突变蛋白。此外，真核基因组内有特定转录错误富集的基因组基因座（TEELs），它们由RNA聚合酶与显著更高的错误率转录并推测为具有影响在癌症，老化和疾病例如唐氏综合征和阿尔茨海默'秒。因此，在遗传学领域对转录错误的研究越来越重要。尽管如此，方法上的障碍限制了准确识别转录错误的进展。Pro-Seq和NET-Seq可以沿基因组纯化新生RNA并绘制RNA聚合酶，但不能用于鉴定转录突变。在这里，我们本背景误差模型耦合的精密核圆形测序上运行（EMPC -SEQ），一种方法COMBIN荷兰国际集团测定和圆形测序核上运行与背景误差模型精确地检测新生转录错误和有效地辨别TEELs基因组中。
[背景]核糖核苷酸错掺导致的转录错误在所有活生物体中无处不在（Carey，2015）。假设每个信使RNA（mRNA）可以翻译2-4千次（Schwanhausser et al。，2011），并且许多特殊RNA在给定时间每个细胞仅表达一次（Islam et al。，2011; Pelechano et al。，2011）。，2010），即使是关键残基的单个转录错误也会使特定蛋白质的表达产生很大差异。另外，转录错误可加速蛋白质聚集，导致人类中与年龄有关的疾病（van ...
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| Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
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Author:
Date:
2020-10-20
[Abstract] Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications ...
[摘要] [摘要] 对DNA结合蛋白和DNA切割蛋白的生化研究通常得益于放射性标记与两个DNA末端之一的特异性连接。在许多情况下，有必要进行两种版本的同一实验：一种是5′DNA末端标记，另一种是3′DNA末端标记。虽然均匀的5′-放射性标记可以通过单个激酶催化磷酸化步骤完成，但现有的3′-放射性标记程序通常会导致探针的异质性，从而妨碍了下游实验中精确的DNA片段鉴定。我们提出了一种新的方案，利用廉价的DNA寡核苷酸修饰（2′-O-甲基核糖核酸和核糖核酸糖取代）、两种酶（T4多核苷酸激酶和T4 RNA连接酶2），将32P磷酸有效地连接到任意序列的DNA寡核苷酸的3′端，以及DNA和RNA对氢氧化物处理的差异敏感性。该方法制备的放射性探针分子具有均一性和氧化剂相容性，可用于DNA酶特性和DNA足迹分析中的精确切割位点定位。
[背景] ...
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