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Molecular and Cellular Biology, November 2000, p. 8432-8446, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Multiple Homing Pathways Used by Yeast Mitochondrial Group II Introns

Robert Eskes,1,dagger Lu Liu,1 Hongwen Ma,2 Michael Y. Chao,1 Lorna Dickson,1,Dagger Alan M. Lambowitz,2 and Philip S. Perlman1,*

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148,1 and Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 787122

Received 16 February 2000/Returned for modification 14 April 2000/Accepted 17 August 2000

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

* Corresponding author. Mailing address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148. Phone: (214) 648-1464. Fax: (214) 648-1488. E-mail: philip.perlman{at}utsouthwestern.edu.

dagger Present address: JWG-Universität Frankfurt am Main ZIM-Med Klinik III-Molekulare Haematologie, D-60596 Frankfurt, Germany.

Dagger Present address: Pacific Northwest Research Institute, Seattle, WA 98122.

Molecular and Cellular Biology, November 2000, p. 8432-8446, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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