Mobility of Yeast Mitochondrial Group II Introns: Engineering a New Site Specificity and Retrohoming via Full Reverse Splicing

• Published in: Cell Vol. 88; no. 6; pp. 865 - 874
• Main Authors: Eskes, Robert, Yang, Jian, Lambowitz, Alan M, Perlman, Philip S
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.03.1997
• Subjects: Base Sequence; DNA, Complementary - analysis; DNA, Complementary - chemical synthesis; DNA, Fungal - analysis; DNA, Mitochondrial - physiology; Endonucleases - genetics; Gene Expression Regulation, Enzymologic - genetics; Gene Expression Regulation, Fungal - genetics; Introns - genetics; Mutagenesis, Site-Directed - genetics; Plasmids; Recombination, Genetic; RNA Splicing - genetics; Saccharomyces cerevisiae; Yeasts - enzymology; Yeasts - genetics
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/S0092-8674(00)81932-7

The mobile group II introns aI1 and aI2 of yeast mtDNA encode endonuclease activities that cleave intronless DNA target sites to initiate mobility by target DNA–primed reverse transcription. For aI2, sense-strand cleavage occurs mainly by a partial reverse splicing reaction, whereas for aI1, complete reverse splicing occurs, leading to insertion of the linear intron RNA into double-stranded DNA. Here, we show that aI1 homing and reverse splicing depend on the EBS1 (RNA)/IBS1(DNA) pairing and that target specificity can be changed by compensatory changes in the target site and the donor intron. Using well-marked strains to follow coconversion of flanking DNA, we show that homing occurs by both RT-dependent and -independent pathways. Remarkably, in most RT- dependent events, the reverse spliced intron is the initial template for first-strand cDNA synthesis.