Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences

Genes Dev. 2000 Mar 1;14(5):559-73.


Group II intron homing occurs primarily by a mechanism in which the intron RNA reverse splices into a DNA target site and is then reverse transcribed by the intron-encoded protein. The DNA target site is recognized by an RNP complex containing the intron-encoded protein and the excised intron RNA. Here, we analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group II intron in vitro and in vivo. Our results suggest a model similar to yeast mtDNA introns, in which the intron-encoded protein first recognizes a small number of nucleotide residues in double-stranded DNA and causes DNA unwinding, enabling the intron RNA to base-pair with the DNA for reverse splicing. Antisense-strand cleavage requires additional interactions between the protein and 3' exon. Key nucleotide residues are recognized directly by the intron-encoded protein independent of sequence context, and there is a stringent requirement for fixed spacing between target site elements recognized by the protein and RNA components of the endonuclease. Experiments with DNA substrates containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in the 3' exon but not the distal 5' exon region. Finally, by applying the target-site recognition rules, we show that the L1.LtrB intron can be modified to insert at new sites in a plasmid-borne thyA gene in Escherichia coli. This strategy should be generally applicable to retargeting group II introns and to delivering foreign sequences to specific sites in heterologous genomes.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Pairing
  • Base Sequence
  • DNA, Antisense / genetics
  • DNA, Antisense / metabolism
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Exons
  • Introns*
  • Lactococcus lactis / genetics*
  • Models, Genetic
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, DNA
  • Templates, Genetic


  • DNA, Antisense
  • DNA, Bacterial