RNA-driven genetic changes in bacteria and in human cells
RNA-driven genetic changes in bacteria and in human cells
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DOI：
10.1016/j.mrfmmm.2011.03.016
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发表时间：
2011-12-01
影响因子：
2.3
通讯作者：
Storici, Francesca
中科院分区：
文献类型：
--
作者：
Shen, Ying;Nandi, Pavan;Storici, Francesca
最近在酿酒酵母模型生物体中使用了合成的含RNA的寡核苷酸，在双链断裂(DSB)修复过程中，RNA可以在染色体水平上作为DNA合成的模板。在这里，我们表明，RNA介导的DNA修饰和修复现象并不局限于酵母细胞。对应于滞后或前导链序列的单链DNA寡聚核苷酸中嵌入的六个核苷酸片段可以作为模板来纠正大肠杆菌染色体中存在缺陷的LacZ标记基因。为了测试RNA在哺乳动物细胞中修饰DNA的能力，我们使用了包含六个核苷酸嵌入链的DNA寡核苷酸，以及主要由RNA组成的寡核苷酸。这些寡核苷酸旨在修复随机整合到人类HEK-293细胞基因组中的绿色荧光蛋白(GFP)基因拷贝中产生的染色体断裂。我们证明了这些含有RNA的寡聚核苷酸可以作为模板来修复人类细胞中的DSB，并可以将碱基变化引入基因组或质粒DNA中。在大肠杆菌和人类细胞中，含有单链RNA的寡核苷酸对染色体基因的纠偏与对相应DNA分子的纠偏是相同的。因此，含有RNA的寡核苷酸在与互补DNA一起退火之前不会转化为cDNA。总体而言，我们证明了在细菌和人类细胞中，就像在酵母中一样，RNA序列可以在DNA遗传修饰和重塑中发挥直接作用。爱思唯尔出版公司(Elsevier B.V.)
As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNA-containing oligonucleotides (oligos), RNA can serve as a template for DNA synthesis at the chromosomal level during the process of double-strand break (DSB) repair. Herein we show that the phenomenon of RNA-mediated DNA modification and repair is not limited to yeast cells. A tract of six ribonucleotides embedded in single-strand DNA oligos corresponding to either lagging or leading strand sequences could serve as a template to correct a defective lacZ marker gene in the chromosome of the bacterium Escherichia coli. In order to test the capacity of RNA to modify DNA in mammalian cells, we utilized DNA oligos containing an embedded tract of six ribonucleotides, as well as oligos mostly made of RNA. These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells. We show that these RNA-containing oligos can serve as templates to repair a DSB in human cells and can introduce base changes into genomic or plasmid DNA. In both E. coli and human cells, the strand bias of chromosomal gene correction by the single-strand RNA-containing oligos was the same as that obtained for the corresponding DNA molecules. Therefore, the RNA-containing oligos are not converted into a cDNA before annealing with complementary DNA. Overall, we demonstrate that in both bacterial and human cells, as in yeast, RNA sequences can have a direct role in DNA genetic modification and remodeling. Published by Elsevier B.V.