Group II intron mobility and its application in gene targeting

The late steps of the group II intron retrohoming require host functions. To identify host factors that are involved in retrohoming, the mobility frequency of the L1.LtrB intron was determined in a number of Escherichia coli mutant strains that are defective in DNA repair. Collaborating with Dr. Belfort's (Albany) group, we have found that the RecJ, RNase Hs, the 5 ' to 3' exonuclease of DNA polymerase I, and DNA polymerase III are likely involved in completion of group II intron retrohoming.; Mobile group II introns use a major retrohoming mechanism in which the intron RNA reverse splices into one strand of a duplex DNA, while the intron-encoded protein site-specifically cleaves the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. By analyzing the mobility mechanism of L1.LtrB intron mutant YRT, which lacks DNA endonuclease activity, I found that the L1.LtrB intron can retrohome in the absence of second-strand cleavage by using a nascent strand generated during target DNA replication as a primer for reverse transcription of the inserted intron RNA. Similar mechanisms may also be used by naturally occurring group II introns that encode proteins lacking the C-terminal DNA endonuclease domain and for the retrotransposition of group II introns to ectopic sites.; Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. I showed that targetrons could be used to disrupt chromosomal genes in E. coli and other bacteria. I also developed a R&barbelow;etrotransposition-A&barbelow;ctivated selectable M&barbelow;arker (RAM), which allows one-step bacterial chromosomal gene disruption at near 100% efficiency after selection. The RAM-containing targetron can be generated rapidly without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. Finally, I showed that a RAM-targetron with randomized target site recognition sequences yields single-insertions throughout the E. coli genome, thus creating a gene knockout library.