Complex recognition site for the group I intron-encoded endonuclease I-SceII.

Abstract:

:We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene. We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site. Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is. Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely. Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs. About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme. I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp. The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate. Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions.

journal_name

Mol Cell Biol

authors

Wernette C,Saldanha R,Smith D,Ming D,Perlman PS,Butow RA

doi

10.1128/mcb.12.2.716

subject

Has Abstract

pub_date

1992-02-01 00:00:00

pages

716-23

issue

2

eissn

0270-7306

issn

1098-5549

journal_volume

12

pub_type

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