Complex recognition site for the group I intron-encoded endonuclease I-SceII.
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.
Wernette
C
C
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Saldanha
R
R
Smith
D
D
Ming
D
D
Perlman
P S
PS
Butow
R A
RA
D001483:Base Sequence; D002893:Chromosomes, Human, Pair 3 / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific