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Bacterial group IIC introns are a subclass of group II intron ribozymes that are typically located downstream from transcriptional terminators. Class IIC- attC  introns constitute a monophyletic subset of subgroup IIC, which preferentially insert into site-specific recombination sequences for integron integrases ( attC ).  attC s are a diverse family of nucleotide sequences composed of conserved inverted repeats that flank a variable, but palindromic, central region. In this study, we used both PCR and colony patch hybridization methods to determine the basis for recognition of the  attC aadA1   stem–loop motif by the  Serratia marcescens  intron ( S.ma .I2) in vivo. The quantitative results showed that mobility into the wild-type site occurs at a frequency of 18%, and is strongly biased by the orientation of the homing site relative to the direction of DNA replication.  S.ma .I2 mobility results into mutant  attC aadA1   sites are consistent with recognition of stem–loop motifs in unwound DNA. The homing frequency results showed that, while the entire  attC  sequence is not necessary for recognition of the insertion site, short deletions of the  attC  stem–loop motif inhibited the intron mobility. Moreover, our data show that  S.ma .I2 requires a bulged base in the folded  attC  stem for high homing frequency. We demonstrate that the IBS1/IBS3 motifs and two bulge bases conserved among  attC s determine  S.ma .I2 homing specificity for the  attC  bottom strand. These results suggest that class IIC- attC  introns tolerate  attC  variation by recognition of a bulged hairpin DNA motif rather than a specific sequence.

Group IIC intron mobility into attC sites involves a bulged DNA stem–loop motif