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

Abstract

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine-or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.Piv catalyzes site-specific inversion of a 2.1-kb chromosomal segment encoding type 4 pilin genes of the human and cow eye pathogens Moraxella lacunata and Moraxella bovis, respectively (14,18,27). This DNA rearrangement results in phase variation of the type 4 pili (19) (Fig. 1). 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).Piv is unique among prokaryotic site-specific DNA invertases because it does not belong to either the tyrosine-or serinerecombinase families. Instead, Piv shares significant amino acid identity and similarity with the transposases of insertion sequences (IS) from the IS110/IS492 family, including three highly conserved amino acid motifs: GDK, KTDDAA, and PSG (14, 24). Piv variants containing alanine or glycine substitutions at multiple positions within each motif are defective for mediating inversion but still bind to the inv and sub sites. The essential role of these conserved motifs in Piv catalysis of DNA inversion suggests that Piv and the transposases of the IS110/IS492 family (Piv/MooV family of DNA recombinases) use similar catalytic mechanisms (25, 31).Most identified transposases belong to the superfamily of retroviral integrases (IN) and are typified by a conserved DDE catalytic motif. The acidic residues of the DDE mot...

Everyman's Guide to Bacterial Insertion Sequences

“…A similar type of Tpase, known as a DEDD Tpase, is related to the Holliday junction resolvase, RuvC (39,40). These possess a similar predicted structural topology in their catalytic site and presumably have similar chemistry to the DDE enzymes.…”

“…At present, only a single IS family, IS110, is known to encode this type of DEDD enzyme, related to the RuvC Holliday junction resolvase (40). There are over 250 examples from nearly 130 bacterial and archaeal species.…”

“…However, the organization of IS110 family members and the inversion systems are different. In the inversion systems, the recombinase is located outside the invertible segment, whereas it is located within the IS element (40).…”

Characterization of thePseudomonas putidaMobile Genetic Element ISPpu10: an Occupant of Repetitive Extragenic Palindromic Sequences

“…After aligning transposases encoded by IS110/IS492 members and proteins encoded by piv genes, Choi and coworkers (8) identified the motif DEDD, relating it to the one found in the catalytic center of the RuvC Holliday junction resolvase. In fact, a model for Piv-mediated inversion that includes resolution of a Holliday junction has been proposed (6).…”

Site-Specific Insertion of IS 492 in Pseudoalteromonas atlantica

“…Transposable elements drive the evolution of genomes through DNA rearrangements resulting from the process of transposition or by homologous recombination between repeated copies of the element in the genome (for a review, see reference 34). Sitespecific recombination systems, which require very short, shared sequences between the recombining DNA segments, often regulate gene expression or generate antigenic diversity (for a review, see reference 16).Specialized DNA recombinases of the DEDD motif family (also called the Piv/MooV family) mediate reactions that have features of both transposition and site-specific recombination (8,18,23,28,29,37). The transposases of the IS110/IS492 family of insertion sequences and the Piv (pilin inversion) sitespecific DNA invertases of Moraxella lacunata and Moraxella bovis are members of the DEDD family of recombinases, (8,23,29,37).…”