Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ∼5–34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection deficiency (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in RPA mutants as well as in rad27Δ and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Together, we propose that a shortage of RPA, typical in cancer cells, may be a factor that stimulates the formation of templated insertions.

中文翻译：

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RPA 和 Rad27 限制了 DNA 断裂处的模板化和反向插入


在 DNA 双链断裂 （DSB） 处形成模板插入在癌细胞中非常常见。调节这些事件的机制和酶在很大程度上是未知的。在这里，我们使用跨修复基因座的扩增子测序研究了 DSB 酵母中的模板化插入。我们在 DSB 记录了非常短 （大多数 ∼5-34 bp） 的模板化反向重复。它们是通过利用与 DSB 相邻的微同源性的折返机制产生的。酶学要求表明一种杂交机制，其中一端需要 Polδ 介导的合成，而另一端被非同源末端连接 （NHEJ） 或选择性末端连接 （Alt-EJ） 捕获。在低水平或突变的 RPA （rtt105Δ;rfa1-t33） 或广泛切除缺陷 （sgs1Δ exo1Δ） 的突变体中，这一过程会加剧。来自不同遥远基因组位置的模板化插入在 RPA 突变体和 rad27Δ 中也有所增加，并且起源于基因组的脆弱区域。在复杂的插入中，常见事件是两个序列的插入，它们起源于同一基因座且方向相反。我们提出这些反转也是由微同源介导的模板切换形成的。总之，我们提出癌细胞中典型的 RPA 短缺可能是刺激模板化插入形成的一个因素。