TAILIEUCHUNG - Báo cáo khoa học: In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional

The AnCOB group I intron fromAspergillus 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 char-acterized the endonuclease activity of I-AniIin vitro and utilizedcompetitionassays toprobe the relationshipbetween theRNA- andDNA-binding sites. | Eur. J. Biochem. 270 1543-1554 2003 FEBS 2003 doi In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein I-AniI William J. Geese Yong K. Kwon Xiaoping Wen and Richard B. Waring Department of Biology Temple University Philadelphia USA 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 stoichio-metrically 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. Keywords Aspergillus nidulans homing endonuclease RNA binding protein DNA sliding RNA splicing. Group I and group II introns frequently contain open reading frames ORFs which are either free-standing within the intron itself or are in-frame with the preceding 5 exon 1 2 . While some of these encode essential host proteins others have been shown to encode proteins that facilitate .

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