Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease
Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease
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DOI：
10.1073/pnas.0810588106
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发表时间：
2009-03-31
影响因子：
11.1
通讯作者：
Stoddard, Barry L.
中科院分区：
文献类型：
--
作者：
Smith, Audrey McConnell;Takeuchi, Ryo;Stoddard, Barry L.
Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wildtype enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at approximate to 1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.
