Efficient DNA double-strand break formation at single or multiple defined sites in the Saccharomyces cerevisiae genome.
Efficient DNA double-strand break formation at single or multiple defined sites in the Saccharomyces cerevisiae genome.
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DOI：
10.1093/nar/gkaa833
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发表时间：
2020-11-18
影响因子：
14.9
通讯作者：
Symington LS
中科院分区：
文献类型：
--
作者：
Gnügge R;Symington LS
DNA 双链断裂 (DSB) 是常见的基因组损伤，威胁基因组稳定性和细胞存活。细胞使用复杂的修复机制来检测和修复 DSB。为了研究 DSB 修复途径和相关因素，诱导型位点特异性核酸内切酶已被证明是基本工具。在酿酒酵母中，半乳糖诱导型稀切核酸内切酶通常用于在独特的切割位点产生单个 DSB。半乳糖诱导需要在次优生长培养基中培养细胞，这非常乏味，尤其是在处理生长缓慢的 DSB 修复突变体时。此外，在酵母基因组的多个确定且独特的位点中同时产生 DSB 的核酸内切酶尚不可用，这阻碍了不同基因组区域和染色质环境中 DSB 修复的研究。在这里，我们提出了克服这些限制的新工具。我们采用异源的不依赖于介质的诱导系统来表达酵母HO核酸内切酶或细菌限制性内切酶，分别用于单个或多个DSB形成。该系统促进在特定基因组位点上严格控制和高效的 DSB 形成，并将成为在局部和全基因组范围内研究 DSB 修复的有价值的工具。
DNA double-strand breaks (DSBs) are common genome lesions that threaten genome stability and cell survival. Cells use sophisticated repair machineries to detect and heal DSBs. To study DSB repair pathways and associated factors, inducible site-specific endonucleases have proven to be fundamental tools. In Saccharomyces cerevisiae, galactose-inducible rare-cutting endonucleases are commonly used to create a single DSB at a unique cleavage site. Galactose induction requires cell cultivation in suboptimal growth media, which is tedious especially when working with slow growing DSB repair mutants. Moreover, endonucleases that simultaneously create DSBs in multiple defined and unique loci of the yeast genome are not available, hindering studies of DSB repair in different genomic regions and chromatin contexts. Here, we present new tools to overcome these limitations. We employ a heterologous media-independent induction system to express the yeast HO endonuclease or bacterial restriction enzymes for single or multiple DSB formation, respectively. The systems facilitate tightly controlled and efficient DSB formation at defined genomic sites and will be valuable tools to study DSB repair at a local and genome-wide scale.
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