Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.
Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.
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使用TrAEL-seq的DNA复制和DNA双链断裂的全基因组分析。
DOI：
10.1371/journal.pbio.3000886
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发表时间：
2021-03
期刊：
影响因子：
9.8
通讯作者：
Houseley J
中科院分区：
文献类型：
--
作者：
Kara N;Krueger F;Rugg-Gunn P;Houseley J
整个基因组的忠实复制需要复制叉来复制大段连续的 DNA，而持续复制叉停滞的位点对基因组稳定性构成了重大威胁。了解复制叉停滞位点的分布以及随后的复制叉处理事件，需要全基因组方法来分析复制叉位置和重组 DNA 末端的形成。在这里，我们描述了转移酶激活末端连接测序 (TrAEL-seq)，这是一种在全基因组范围内捕获单链 DNA 3' 末端并具有碱基对分辨率的方法。 TrAEL-seq 标记 DNA 断裂和复制叉，提供酵母和哺乳动物细胞中复制叉进展和复制叉停滞位点的全基因组图谱。复制图谱与冈崎片段测序得到的相似；然而，TrAEL-seq 是在野生型细胞的异步群体上进行的，无需加入标签、细胞分选或复制中间体的生化纯化，这使得 TrAEL-seq 比现有的复制叉方向分析方法更简单、更广泛适用。 TrAEL-seq 对 DNA 3' 末端的特异性还允许在 DNA 末端切除开始后准确检测双链断裂位点，我们通过对 dmc1Δ 突变体中减数分裂双链断裂热点的全基因组作图证明了这一点，该突变体能够进行末端切除但不能进行链入侵。总体而言，TrAEL-seq 为研究 DNA 复制和修复提供了一种灵活而稳健的方法，具有高灵敏度和分辨率，这对于确定基因组不稳定机制具有重要作用。 TrAEL-seq 提供暴露 DNA 3' 末端的全基因组碱基对分辨率图；这揭示了异步、未标记的野生型细胞群中的复制叉停滞和正常复制特征，以及切除的 DNA 断裂位点。
Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability. TrAEL-seq provides genome-wide base pair resolution maps of exposed DNA 3’ ends; this reveals replication fork stalling and normal replication profiles in asynchronous, unlabelled wildtype cell populations, along with the sites of resected DNA breaks.
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