Enrichment of DNA replication intermediates by EdU pull down.

4区生物学 Q4 Biochemistry, Genetics and Molecular Biology

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-12-13 DOI:10.1016/bs.mcb.2022.11.001

Fabio Pessina, Alessia Romussi, Daniele Piccini, Giulia Mazzucco, Mario Varasi, Ylli Doksani

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引用次数: 0

Abstract

Analysis of replication fork structures in electron microscopy (EM) can provide important mechanistic insights in DNA replication studies. A major challenge in this type of analysis is the paucity of replication intermediates. At any given time only a small fraction of the restriction fragments of genomic DNA will contain a replication fork. To address this issue, we have developed an EdU-pull-down procedure to enrich for replicating DNA. Cells are exposed to a brief pulse of EdU, a cleavable biotin moiety is attached to EdU by copper-catalyzed azide-alkyne cycloaddition (CuAAC), in conditions that minimize the damage to DNA. Biotinylated DNA is purified with streptavidin beads, in conditions that facilitate association of long DNA filaments. Finally, the DNA is eluted by cleaving the biotin moiety. This approach can enrich over 150-times for replicating DNA and about 50-times in replication fork structures, as verified by EM. This procedure could benefit analysis of replication intermediates in EM as well as other techniques for the study of replicating DNA.

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通过 EdU 牵引富集 DNA 复制中间体。

用电子显微镜（EM）分析复制叉结构可为 DNA 复制研究提供重要的机理启示。这类分析的一个主要挑战是复制中间体太少。在任何时候，基因组 DNA 的限制性片段中只有一小部分含有复制叉。为了解决这个问题，我们开发了一种 EdU 下拉程序来富集复制 DNA。将细胞暴露于短暂的 EdU 脉冲，通过铜催化的叠氮-炔环加成（CuAAC）将可裂解的生物素分子连接到 EdU 上，条件是尽量减少对 DNA 的损伤。用链霉亲和素珠子纯化生物素化的 DNA，纯化条件要有利于长 DNA 细丝的结合。最后，通过裂解生物素分子洗脱 DNA。经 EM 验证，这种方法可富集复制 DNA 150 倍以上，富集复制叉结构约 50 倍。这种方法有利于在 EM 中分析复制中间体，也有利于利用其他技术研究复制 DNA。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in cell biology 生物-细胞生物学

CiteScore

3.10

自引率

0.00%

发文量

125

审稿时长

3 months

期刊介绍： For over fifty years, Methods in Cell Biology has helped researchers answer the question "What method should I use to study this cell biology problem?" Edited by leaders in the field, each thematic volume provides proven, state-of-art techniques, along with relevant historical background and theory, to aid researchers in efficient design and effective implementation of experimental methodologies. Over its many years of publication, Methods in Cell Biology has built up a deep library of biological methods to study model developmental organisms, organelles and cell systems, as well as comprehensive coverage of microscopy and other analytical approaches.