期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 13, 页码 5099-5104出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0810588106
关键词
protein engineering; recombination; single strand breaks; gene therapy; gene repair
资金
- National Institutes of Health [R01 GM49857, RL1 CA133833, 1RL1 CA133831, RL1 GM084434]
- Gates Foundation
- National Science Foundation
- Japan Society for the Promotion of Science
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.
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