Yu Yang, Wang Xin, Fox Jordan, Li Qian, Yu Yang, Hastings P J, Chen Kaifu, Ira Grzegorz
Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA.
Department of Cardiology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1159.
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.
在癌细胞中，DNA双链断裂（DSB）处模板化插入的形成非常普遍。调节这些事件的机制和酶在很大程度上尚不清楚。在这里，我们使用跨修复位点的扩增子测序研究了酵母中DSB处的模板化插入。我们记录了DSB处非常短（大多数约5 - 34个碱基对）的模板化反向重复序列。它们是通过一种利用与DSB相邻的微同源性的回折机制产生的。酶学要求表明这是一种混合机制，其中一端需要Polδ介导的合成，而另一端则通过非同源末端连接（NHEJ）或替代末端连接（Alt-EJ）捕获。在RPA水平低或发生突变的突变体（rtt105Δ；rfa1-t33）或广泛切除缺陷的突变体（sgs1Δ exo1Δ）中，这个过程会加剧。来自各种遥远基因组位置的模板化插入在RPA突变体以及rad27Δ中也会增加，并且起源于基因组的脆弱区域。在复杂插入中，常见的事件是两个序列的插入，它们来自同一基因座且方向相反。我们提出这些倒位也是由微同源性介导的模板切换形成的。总之，我们认为癌细胞中典型的RPA短缺可能是刺激模板化插入形成的一个因素。
