Multiple DNA Binding Activities of the Novel Site-specific Recombinase, Piv, from Moraxella lacunata

Abstract

The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/ IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.Numerous site-specific DNA recombination systems and DNA transposition systems have been characterized biochemically and have been found to follow two distinct chemical pathways for DNA cleavage and strand transfer in recombination (reviewed in Refs. 1-4). Site-specific recombination, mediated by the recombinases of the -integrase and Hin/resolvase families, involves a two-step transesterification reaction in which the intermediate is a covalent recombinase-DNA linkage. This covalent attachment is the result of nucleophilic attack on the DNA phosphodiester backbone by a hydroxyl group of the conserved serine (Hin/resolvase), or tyrosine (-integrase), of the recombinase. In the second transesterification reaction, the phosphodiester linkages of the exchanged DNA strands are restored (reviewed in Refs. 2 and 3). In contrast, DNA transposition, mediated by transposases containing the catalytic DDE amino acid motif, utilizes a hydrolysis reaction for cleavage at the ends of the transposable element. This first cleavage leaves 3Ј-OH ends to act directly as the attacking nucleophile in a one-step trans-esterification reaction resulting in strand exchange. Resolution of the transposition process involves DNA replication or DNA repair activity to fill in gaps left at the target site due to the staggered cut mediated by the transposase and the 3Ј hydroxyl groups at the element ends (reviewed in Refs. 1 and 4).These features of the recombination reactions mediated by site-specific recombinases and transposases suggest that a group of related recombinases would not mediate both sitespecific recombination and transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DN...

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

“…The invertase Piv, encoded immediately adjacent to the invertible segment, is expressed from P piv (10). sub1 is a nonessential Piv binding site (32). for 45 s, and 1 cycle at 72°C for 7 min) was electrophoresed on a 1.2% agarose gel and visualized as described above.…”

“…The invertible segment is bounded by identical 32-bp inverted repeats, invL and invR, where Piv mediates recombination. In addition, Piv interacts with an accessory site, sub1, which may facilitate assembly of an active synaptic complex (32).…”

Analysis of the Piv Recombinase-Related Gene Family of Neisseria gonorrhoeae

“…Neither of the two homologues could complement Piv or MooV activities in these systems. While there are a number of possible reasons why complementation was not seen, it is most likely due to differences in DNA binding or cleavage site specificity rather than catalytic activity; Piv and MooV cannot complement for each other, as well (41). There is very little similarity between the Piv-binding sites within the inversion region of M. lacunata and the flanking sequences of the irg genes (data not shown), and the sites for MooV binding and DNA cleavage on IS492 have not yet been determined.…”

“…irg2 (pAG712) and irg7 (pAG711) were amplified with Pfu polymerase (Promega) by using primer pairs 5Ј-CAGTTCAGCTAGCCGT AACGCCGTAGGATTGG-3Ј and 5Ј-CTCGTCTCGAGCGCCGATTTGTAA CGCGATGG-3Ј as well as 5Ј-CAGTTCACATATGAATATAATCGGGCCGG ACATC-3Ј and 5Ј-CTCGTCTCGAGTTGATTCAATCGGTGTCTTTCC-3Ј introduced into the NheI/XhoI and NdeI/XhoI sites, respectively, of pET21a (Novagen) and expressed from the T7 promoter as His 6 -tagged proteins in E. coli HMS174(DE3). The pivML (pAG1300) and mooV (pAG921) expression vectors, containing the recombinase genes in pET21a between the NdeI/XhoI sites, have previously been shown to exhibit in vivo inversion and precise excision activities, respectively (21,41). Recombinase expression was induced with 0.2 mM IPTG, and samples were taken at 2 h postinduction from both induced and uninduced cultures for Western blot analysis as described previously by Tobiason et al (40).…”

The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1

“…This region of the ADP1 chromosome regulates the catabolism of p-hydroxybenzoate (DiMarco et al, 1993) and does not contain a recognizable binding sequence for CatM or BenM. Competition studies were done by adding an increasing range of competitor DNA, from 1-to 50-fold molar excess, to the reaction as previously described (Tobiason et al, 1999).…”