Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting.
Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting.
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DOI：
10.1016/j.dnarep.2014.07.004
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发表时间：
2014-10
期刊：
影响因子：
3.8
通讯作者：
Svetec IK
中科院分区：
文献类型：
--
作者：
Štafa A;Miklenić M;Zunar B;Lisnić B;Symington LS;Svetec IK
基因打靶在酿酒酵母中是非常有效的。它是通过与线性的、非复制的DNA片段进行转化来执行的，该DNA片段携带可选择的标记并包含与基因组中特定基因座同源的末端。然而，即使在酿酒酵母中，转化也会导致不想要的(异常的)整合事件，其频率和光谱对于末端转化和末端转化分析来说是非常不同的。已经观察到，基因替换(末端基因打靶)可以导致不合法的整合、目标序列旁边的转化DNA片段的整合和目标染色体的复制。相比之下，质粒整合(末端基因打靶)通常与多个靶向整合事件有关，但非法整合极其罕见，且尚未有靶向染色体复制的报道。在这里，我们系统地研究了末端剔除试验的设计对靶向基因修饰成功的影响。我们已经确定了几种末端基因打靶试验中的转化效率、基因打靶的保真度和所有异常事件的光谱，这些分析旨在插入、删除或替换酵母基因组目标区域中的特定序列。此外，我们首次证明，即使在末端基因打靶的过程中，也会发生有针对性的染色体复制。最重要的是，整个染色体复制是POL32依赖的，表明断裂诱导复制(BIR)是潜在的机制。此外，外显子1Δ-sgs1-Δ双突变体显著增加了靶染色体复制的发生，而在各自的单个突变体中则没有增加，说明外显子1和sgs1蛋白在基因打靶过程中独立地抑制了整个染色体的复制。
Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.
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