Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy.
Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy.
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DOI：
10.1007/978-1-0716-2477-7_6
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发表时间：
2022
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DNA - rna杂交可以干扰DNA复制，但潜在的中间体和分子机制仍然难以捉摸。在这里，我们描述了一种单分子方法，可以在持续复制的背景下监测DNA-RNA杂交的特异性位点。使用限制性酶切，凝胶电泳和凝胶洗脱，该工作流程可以有效地分离复制中间体，并研究特定基因组位点的复制动力学。在这里，我们应用这个程序来分离携带诱导转录-复制冲突的细菌基因组位点。此外，我们结合电子显微镜和S9.6-Gold免疫标记检测分离的复制中间体上的DNA-RNA杂交体。虽然有一些局限性，但这种方法可以适用于不同生物体的基因座特异性复制分析。
DNA–RNA hybrids can interfere with DNA replication, but the underlying intermediates and molecular mechanisms have remained elusive. Here, we describe a single molecule approach that allows to monitor DNA–RNA hybrids locus-specifically in the context of ongoing replication. Using restriction digestion, gel electrophoresis and gel elution, this workflow allows to efficiently isolate replication intermediates and to study replication dynamics across a specific genomic locus. Here, we applied this procedure to isolate a bacterial genomic locus carrying an inducible transcription-replication conflict. Moreover, we combined electron microscopy with S9.6-Gold immuno-labeling to detect DNA–RNA hybrids on the isolated replication intermediates. With some limitations, this approach may be adapted to locus-specific replication analyses in different organisms.