Developing a programmed restriction endonuclease for highly specific DNA cleavage
Developing a programmed restriction endonuclease for highly specific DNA cleavage
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DOI：
10.1093/nar/gki1009
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Pingoud, A
中科院分区：
文献类型：
--
作者：
Eisenschmidt, K;Lanio, T;Pingoud, A
大DNA分子在少数位点的特异性切割是分析基因组DNA或靶向复杂基因组中的单个基因所必需的，需要具有极高特异性的核酸内切酶。识别4- 8bp的DNA序列的限制性内切核酸酶（REase）对于该目的没有足够的特异性。原则上，REase的特异性可以通过与序列识别模块融合来延伸，例如特异性DNA结合结构域或三螺旋形成寡核苷酸（TFO）。我们已经选择使用TFO来扩展REases的特异性，考虑到这种融合提供的组合灵活性，其能够解决紧挨着定义的三螺旋形成位点（TFS）的短的但精确识别的限制性位点。我们在这里证明了PvuII的单链变体（scPvuII）通过双功能交联剂N-（γ-马来酰亚胺基丁氧基）琥珀酰亚胺酯转化为TFO（5 ′-NH 2-[CH 2]（6或12）-MPMPMPMPMPPPPPPT-3 ′，其中M是5-甲基-2 ′-脱氧胞苷，P是5-[1-丙炔基]-2 ′-脱氧尿苷），如果位于距TFS约一个螺旋转角的距离，则在PvuII的识别位点（CAGCTG）特异性切割DNA（下划线）与TFO互补（“寻址”位点：5“-（类似于10）CAGCTG-3”），留下“未寻址”PvuII位点完整。如果在不存在Mg 2+离子的情况下通过在scPvuII-TFO和底物预孵育之后添加Mg 2+离子来引发切割反应以允许在DNA切割之前形成三螺旋，则与"未寻址“位点相比，”寻址“位点的切割优先性> 1000倍。TFS中的单碱基对取代阻止了scPvuII-TFO对DNA的切割。
Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-(similar to 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is > 1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.