Open AccessIn vitro analysis of the relationship between endonuclease and maturase activities in the bi‐functional group I intron‐encoded protein, I‐AniI
Author(s) -
Geese William J.,
Kwon Yong K.,
Wen Xiaoping,
Waring Richard B.
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03518.x
Subject(s) - homing endonuclease , endonuclease , rna , intron , nucleic acid , dna , biology , rna splicing , biochemistry , microbiology and biotechnology , gene
The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I‐AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I‐AniI in vitro and utilized competition assays to probe the relationship between the RNA‐ and DNA‐binding sites. Despite functioning as an RNA maturase, I‐AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA‐binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I‐AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I‐AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I‐AniI appears to contain two binding sites.