The DNA-binding and DNA endonuclease domains of a group II intron-encoded protein: characterization and application to the engineering of gene-targeting vectors

Group II introns are both catalytic RNAs and mobile genetic elements that insert site-specifically into intronless alleles, a process termed retrohoming. Retrohoming occurs by a mechanism in which the excised intron RNA inserts directly into one strand of the DNA, while the intron-encoded protein (IEP) cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. Both the IEP and intron RNA base pairing contribute to DNA target site recognition and re-scripting the intron RNA allows homing into new sites. My objective was to investigate interactions between the IEP and DNA, with emphasis on identifying critical protein regions, amino acid residues, and mechanisms of recognition and cleavage of the target DNA. First, by using an E.coli genetic system, I selected variants of the Lactococcus lactis Ll.LtrB intron that insert into heterologous chromosomal DNA target sites in L.