期刊
NUCLEIC ACIDS RESEARCH
卷 37, 期 3, 页码 877-890出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkn1007
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资金
- NCI NIH HHS [RL1 CA133833, RL1 CA133832] Funding Source: Medline
- NIGMS NIH HHS [T32 GM07270, R01 GM049857, R01 GM49857] Funding Source: Medline
The LAGLIDADG homing endonuclease (LHE) I-Anil has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-Anil. This implies that mutations that have previously reduced the endonuclease activity of I-Anil are identified and reversed, sometimes in combination with additional 'artificial' mutations, to optimize its in vivo activity.
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