Structural and functional analysis of a novel recombinase
Buchner, John Michael
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The DNA site-specific invertase Piv catalyzes the inversion of a 2.1 kb segment in Moraxella lacunata and Moraxella bovis altering the expression of type 4 pilin genes in two phases. The switch is a biphasic system, alternating between expression the tfpI and tfpQ pilin genes. In M. lacunata this switch is an on/off phase variation. Outside but adjacent to the invertible region, piv encodes the DNA invertase (Piv) that interacts with invL and invR as the loci of recombination. Other sites of Piv DNA binding have been observed, and one, sub1, has been characterized. Piv does not have homology with the traditional site-specific tyrosine or serine recombinases. Instead, Piv has homology to the transposases of the IS110/IS492 family of insertion elements. Modeling of Piv revealed three structures of interest: 1) the ribonuclease H-like fold, a catalytic domain associated with DDE-motif transposases, retroviral integrases, and RuvC Holliday junction resolvases, 2) a potential leucine zipper that could be the site of protein dimerization, 3) a predicted helix-hairpin-helix (HhH) DNA binding motif, best studied in RuvA, a protein that binds Holliday junctions in a non sequence-specific manner. We determined that four acidic residues that are conserved among Piv and the recombinases of the IS110/IS492 family of recombinases are required for catalysis of inversion and comprise a DEDD motif in Piv like that of RuvC. There was no requirement for the predicted leucine zipper in our in vivo inversion system. We also mutated residues that are conserved in a consensus HhH sequence and observed that they are required for DNA inversion and may play a role in binding to Holliday junctions in vitro. Through electrophoretic mobility shift assays, we observed an affinity of Piv for Holliday junctions and branched DNA substrates containing mismatched DNA. This leads us to our hypothesis: Piv is unique among enzymes in that it is a site-specific invertase that catalyzes recombination through hydrolysis and transesterification, like the DDE-motif transposases, and generates a Holliday junction intermediate that is resolved by Piv with a pair of hydrolysis reactions, like RuvC.