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.

FIG. 1.

Inversion region on the M. lacunata chromosome. Recombination sites invL and invR are within the coding sequence of the type 4 pilin genes tfpQ and tfpI such that inversion switches the 3′ coding sequence of the gene expressed from P_tfp. M. bovis alternately expresses the serologically different pilins, but M. lacunata exhibits a on-off phase variation of TfpQ pili due to a frameshifting 19-bp duplication (black box) in tfpI (18). The invertase Piv, encoded immediately adjacent to the invertible segment, is expressed from P_piv (10). sub1 is a nonessential Piv binding site (32).

FIG. 2.

Substitutions in Piv. The predicted catalytic residues within the amino terminal 160 amino acids of Piv are highlighted by gray boxes, and the nonconserved glutamic acid residues that were also targeted for mutagenesis are in open boxes. The substituted residues are indicated in gray type below the wild-type amino acid.

FIG. 3.

Expression of Piv variants in the strain used for inversion assays. Mid-log cultures of DH5α, or DH5α carrying pAG800.2 encoding the wild-type (wt) or mutated piv genes (the substitutions are indicated above each lane) were induced with 100 μM IPTG, and at 2 h postinduction, cells were harvested and lysed and the proteins were fractionated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels. The Western blot of this gel, utilizing anti-Piv antisera as primary antibody, is shown. Piv is marked by an arrow.

FIG. 4.

In vivo inversion activity of Piv variants substituted within the DEDD motif and at nonconserved glutamate residues. DH5α, containing the inversion substrate pMxL90, was transformed with pAG800.2-derived expression vectors, encoding wild-type Piv or the variants with the indicated substitutions, and Piv expression was induced with 50 μM IPTG. Plasmid DNA was isolated from overnight cultures of individual transformants, and inversion of the type 4 pilin segment on pMxL90 was determined by digestion with BsrGI. Digest products were electrophoresed on a 0.6% agarose gel and stained with EtBr (inverted image is shown). The starting Q orientation of the invertible segment on pMxL90 (Q) yields unique 6.5- and 2.6-kb fragments (pMxL90 alone); inversion to the I orientation gives unique 5.2- and 3.9-kb fragments (pMxL100 alone); a 2.5-kb fragment is common to both. The expression vectors contain only one BsrGI site, giving a single 7-kb fragment (pAG800.2 alone).

FIG. 5.

In vivo intermolecular recombination mediated by Piv variants substituted at acidic residues. DNA from the inversion assays with Piv variants described in Fig. 4 was assayed for intermolecular recombination between invL on pMxL90 and invR on the pAG800.2-derived vectors. The new DNA junction was detected by PCR using primers, designated P1 and P2, that anneal to sequence unique to pAG800.2 and pMxL90, respectively. PCR products were electrophoresed on a 1.2% agarose gel and stained with EtBr (inverted image is shown). The 1,073-bp PCR product was sequenced from selected reactions to confirm that recombination occurred within the inv sequences.

FIG. 6.

Detection of low level in vivo inversion activity of Piv variants. DNA template utilized in the recombination assays described in Fig. 4 and 5 was used in a three-primer PCR inversion assay. A primer that anneals to tfpB sequence on pMxL90 pairs with one of two primers that anneal to sequence flanking the invertible segment to yield a 981- or a 811-bp PCR product when the invertible segment is in the “Q” or “I” orientation, respectively. The PCR products were electrophoresed on a 1.2% agarose gel and stained with EtBr (inverted image is shown).

FIG. 7.

In vivo DNA binding activity of Piv variants. The invL recombination site, introduced at the +1 position relative to a constitutive promoter, acts as an operator sequence controlling expression of lacZ on a single-copy plasmid, pAR110 (7). Piv wild-type (wt) and Piv variant (substitutions are indicated) expression vectors were transformed into the DH5α strain containing pAR110, and the transformants were assayed for β-galactosidase activity. The β-galactosidase activity is indicated as Miller units per μg protein.

FIG. 8.

A working model for Piv-mediated site-specific DNA inversion. The synapsed invL (black lines) and invR (gray lines) are shown bound by a Piv dimer (gray circles). Piv mediated-DNA hydrolysis at one recombination site (depicted as invL) (a) and strand transfer (b) lead to formation of a Holliday junction structure. Repositioned Piv catalytic sites now cleave the outer strands of the junction (c), and host DNA ligase activity repairs the nicks (d).