Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage
Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage
复制标题
DOI：
10.1101/gr.109033.110
复制
发表时间：
2011-03-01
期刊：
影响因子：
7
通讯作者：
Rothstein, Rodney
中科院分区：
文献类型：
--
作者：
Reid, Robert J. D.;Gonzalez-Barrera, Sergio;Rothstein, Rodney
我们通过开发一种新的基于交配的高通量方法，称为选择性倍性消除（SPA），简化了将质粒转移到任何酵母菌株文库中的过程。SPA使用在每条染色体上含有条件着丝粒的通用质粒供体菌株。将携带质粒的供体与受体交配，然后去除所有供体菌株染色体，产生含有转移质粒的单倍体菌株。作为原理的证明，我们使用SPA将含有DNA拓扑异构酶I（TOP 1）的野生型和突变型等位基因的质粒转移到单倍体酵母基因破坏文库中。Top1的过表达仅鉴定了一个敏感突变rpa 34，而top1-T（722）A等位基因（一种喜树碱模拟物）的过表达鉴定了190个敏感的基因破坏菌株沿着rpa 34。除了已知的喜树碱敏感菌株，这套包含在Rpd 3组蛋白去乙酰化酶复合物，动粒，和囊泡运输基因的突变。我们进一步表明，在几个ESCRT囊泡运输组件的突变增加Top1水平，这是依赖于SUMO修饰。这些发现证明了SPA技术将质粒引入到单倍体基因破坏文库中以发现新的相互作用途径的实用性。
We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T(722)A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.