Department of Molecular Genetics and Microbiology and the University Program in Genetics and Genomics, Duke University, Durham, North Carolina, United States of America.
PLoS Genet. 2018 Mar 26;14(3):e1007302. doi: 10.1371/journal.pgen.1007302. eCollection 2018 Mar.
Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast.
有丝分裂重组可导致杂合性丢失和染色体重排，从而塑造基因组结构并引发人类疾病。 工程双链断裂（DSB）是重组的有效启动子，但自发事件是否从一条或两条 DNA 链的断裂开始尚不清楚。 在本研究中，使用交叉（CO）特异性测定法比较了异源双链 DNA（hetDNA）谱，该谱反映了链交换中间体，与酵母中 DSB 诱导的事件与自发事件相关。 大多数 DSB 诱导的 CO 产物具有经典 DSB 修复模型预测的双侧 hetDNA，hetDNA 位置在断裂处从一个产物切换到另一个产物。 然而，大约 40％的 CO 仅在起始断裂的一侧具有 hetDNA。 这种异常可以通过一种修改后的模型来解释，其中在延伸入侵末端之前频繁处理早期入侵（D 环）中间体。 最后，hetDNA 片段表现出与 DSB 频繁扩展为缺口、链交换结迁移以及在缺口填充反应期间模板转换一致的复杂性。 在野生型背景或活性氧水平升高的背景（tsa1Δ 突变体）中分离的自发 CO 中的 hetDNA 模式与与 DSB 诱导事件相关的模式相似，表明 DSB 是酵母中自发有丝分裂重组的主要启动子。
