INVIVO AND INVITRO ANALYSES OF AN INTRON-ENCODED DNA ENDONUCLEASE FROM YEAST MITOCHONDRIA - RECOGNITION SITE BY SITE-DIRECTED MUTAGENESIS

被引:36
作者
SARGUEIL, B
HATAT, D
DELAHODDE, A
JACQ, C
机构
[1] Laboratoire de Génétique Moléculaire, CNRS UA 1302, Ecole Normale Supérieure, 75230 Paris Cedex 05
关键词
D O I
10.1093/nar/18.19.5659
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The pal 4 nuclease (termed I-Sce II) is encoded in the group I al 4 intron of the COX I gene of Saccharomyces cerevisiae. It introduces a specific double-strand break at the junction of the two exons A4-A5 and thus mediates the insertion of the intron into an intronless strain. To define the sequence recognized by pal 4 we introduced 35 single mutations in its target sequence and examined their cleavage properties either in vivo in E. coli (when different forms of the pal 4 proteins were artificially produced) or in vitro with mitochondrial extracts of a mutant yeast strain blocked in the splicing of the al 4 intron. We also detected the pal 4 DNA endonuclease activity in extracts of the wild type strain. The results suggest that 6 to 9 noncontiguous bases. In the 17 base-pair region examined are necessary for pal 4 nuclease to bind and cleave its recognition site. We observed that the pal 4 nuclease specificity can be significantly different with the different forms of the protein thus explaining why only some forms are highly toxic in E. coli. This study shows that pal 4 recognition site is a complex phenomenon and this might have evolutionary implications on the transfer properties of the intron. © 1990 Oxford University Press.
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页码:5659 / 5665
页数:7
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