The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens.
The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens.
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DOI：
10.1371/journal.pone.0161204
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Cuming AC
中科院分区：
文献类型：
--
作者：
Kamisugi Y;Whitaker JW;Cuming AC
模式苔藓植物小立壶菌（Physcomitrella patens）是植物中唯一支持易同源重组介导的基因靶向（easy homologous recombin- mediated gene targeting， GT）产生突变等位基因的植物。考虑到靶向转基因整合是通过同源依赖的DNA双链断裂（DNA- dsb）修复途径捕获转化DNA发生的，我们分析了对博莱霉素诱导的DNA损伤的全基因组转录组反应，并在候选DNA修复基因中产生突变。大规模平行（Illumina） cDNA测序确定了基因靶向的潜在参与者。在多种修复途径中活跃的DNA修复蛋白编码转录物显著上调。其中包括同源依赖性修复中的Rad51、CtIP、DNA连接酶1、复制蛋白A和ATR，非同源末端连接中的Xrcc4、DNA连接酶4、Ku70和Ku80以及微同源介导的末端连接中的Rad1、Tebichi/聚合酶theta、PARP。差异调节的细胞周期成分包括上调的Rad9和Hus1 dna损伤相关检查点蛋白和下调的d型细胞周期蛋白和b型CDKs，与同源依赖性DNA-DSB修复细胞周期特征的G2检查点的施加相称。候选基因，包括与修复和重组相关的atp依赖性染色质重塑解旋酶，被敲除并分析生长缺陷、对DNA损伤的超敏感性和GT效率降低。PpCtIP是一种细胞周期激活的同源依赖性DSB切除介质，靶向敲除PpCtIP会导致博莱霉素过敏，并大大降低GT效率。
The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.