Content area
Abstract
Piv, the site-specific recombinase in the Moraxella DNA inversion system, is proposed to utilize a unique mechanism for recombination due to its significant amino acid homology with the transposases of the IS110/IS492 family of insertion elements. This study examines Piv's DNA-binding activity as well as the function of several amino acid motifs, which are conserved between Piv and these transposases. Binding assays revealed that Piv has two distinct DNA recognition sites, sub and inv. The site of inversion, inv , is located at the ends of the invertible segment, while the sub1 site is located 265 bp upstream of the invL site. Additional sub-like sites were also identified within the inversion region. Piv has a high affinity for the sub1 site to which one or more Piv protomers, bind. The results suggest that one Piv protomer binds to this site and via cooperative interactions a second Piv protomer is bound, possibly forming an oligomer. Piv has a much lower affinity for the inv site and only binds to this site as a multimer. A model is proposed for the role that these Piv DNA recognition sites may play in the inversion reaction.
Site-directed mutagenesis was performed on three conserved amino acid motifs, previously identified between Piv and the transposases, of the IS 110/IS492 family. These Piv mutants were tested for their inversion and DNA-binding activity in vivo. All of these mutations destroyed Piv's ability to invert Moraxella DNA. However, these mutants were still able to bind to both the sub and inv sites in vivo, suggesting that these conserved residues have a catalytic role in the inversion reaction. An additional mutation was made at two non-conserved serines which had no effect upon either Piv's inversion or DNA-binding activity.
The results presented here suggest that Piv is a novel recombinase which utilizes a mechanism for site-specific DNA recombination distinct from any of the recombination/transposition systems previously studied.
