Eskes R, Liu L, Ma H, Chao M Y, Dickson L, Lambowitz A M, Perlman P S
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.
Mol Cell Biol. 2000 Nov;20(22):8432-46. doi: 10.1128/MCB.20.22.8432-8446.2000.
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.
酵母线粒体DNA II类内含子aI1和aI2是逆转录元件，它们通过一种称为归巢的过程特异性地插入无内含子的等位基因位点。在这里，我们利用与野生型和突变型aI2内含子杂交时侧翼标记共转化的模式，区分出三种共存的归巢途径：两种依赖逆转录酶（RT）（逆转录归巢），一种不依赖RT。所有这三种途径都是由内含子编码的内切核酸酶切割受体DNA靶位点启动的，有义链通过部分或完全反向剪接被切割，反义链由内含子编码的蛋白质切割。标准杂交中的主要逆转录归巢途径导致内含子插入，上游外显子序列单向共转化。这种共转化模式表明，主要的逆转录归巢途径是由反向剪接的内含子RNA的靶DNA引发的逆转录启动的，并通过与供体等位基因的双链断裂修复（DSBR）重组完成。不依赖RT的途径导致内含子插入，双向共转化，推测是由内含子编码的内切核酸酶切割受体DNA靶位点引发的传统DSBR重组机制发生的，就像I类内含子归巢一样。最后，一些突变的DNA靶位点将高达43%的逆转录归巢转移到另一条以前未在aI2中检测到的途径，其中侧翼外显子序列没有共转化。这条新途径可能涉及插入的内含子RNA全长cDNA拷贝的合成，并通过独立于同源重组的修复过程完成，就像乳酸乳球菌Ll.LtrB内含子那样。我们的结果表明，II类内含子的移动性可以通过多种途径发生，其比例取决于内含子和DNA靶位点的特征。这种显著的灵活性使II类内含子能够在不同的宿主细胞中使用不同的重组和修复酶。
