Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI
Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI
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DOI：
10.1006/bbrc.1999.0152
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发表时间：
1999-02-05
影响因子：
3.1
通讯作者：
Cantrell, MA
中科院分区：
文献类型：
--
作者：
Monnat, RJ;Hackmann, AFM;Cantrell, MA
我们已经确定了两个很好的特点真核归巢核酸内切酶，I-PpoI从粘菌多头绒泡菌和I-CreI从绿色衣藻reinhardtii，在人类细胞中产生位点特异性DNA双链断裂的能力。这些18-kDa蛋白质在它们各自的宿主中切割高度保守的15-或24-bp rDNA归巢位点以产生启动靶内含子转座或“归巢”的均质4-碱基3'末端。“我们表明这两种核酸内切酶都可以在人类细胞中表达，并且可以在28 S rDNA和归巢位点质粒中产生位点特异性DNA双链断裂。这些核酸内切酶诱导的断裂可以在体内修复，尽管断裂修复是致突变的，经常产生短缺失或插入。I-PpoI和I-CreI可用于分析人类细胞和rDNA中的DNA双链断裂修复。(C)北京：科学出版社.
We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycete Physarum polycephalum and I-CreI from the green alga Chlamydomonas reinhardtii, to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3' ends that initiate target intron transposition or "homing." We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repaired in vivo, although break, repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA. (C) 1999 Academic Press.