Genome-wide requirements for resistance to functionally distinct DNA-damaging agents
Genome-wide requirements for resistance to functionally distinct DNA-damaging agents
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DOI：
10.1371/journal.pgen.0010024
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发表时间：
2005-08-01
期刊：
影响因子：
4.5
通讯作者：
Giaever, G
中科院分区：
文献类型：
--
作者：
Lee, W;Onge, RPS;Giaever, G
尽管进行了大量的研究，但细胞对dna损伤化合物反应的机制和治疗差异仍未完全了解。为了扩大我们对DNA损伤的认识，我们分析了12种密切相关的DNA损伤剂对4,700个类似条形码纯合缺失菌株的完整库的影响。在我们的方案中，缺失菌株被集中在一起，在化合物的存在下竞争性地生长。相对应变敏感性是通过pcr扩增的条形码与携带条形码补体的寡核苷酸阵列杂交来确定的。这些筛选鉴定了具有良好特征的dna损伤反应途径中的基因，以及在dna损伤反应中未被确定作用的基因。高通量个体生长分析用于独立确认微阵列结果。每种化合物都产生了独特的全基因组图谱。对这些数据的分析使我们能够确定dna修复模块对12种化合物抗性的相对重要性。对12种不同化合物的数据进行聚类，揭示了构成dna损伤反应的已知和新的功能相互作用，并使我们能够定义修复链间交联所需的遗传决定因素。进一步的遗传分析可以确定其中一个功能群的上位性。
The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of similar to 4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.