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 inMoraxella 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 λ 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 ofM. 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. The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment inMoraxella 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 λ 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 ofM. 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. isopropyl-1-β-d-galactopyranoside polymerase chain reaction base pair(s) kilkobase pair(s) maltose-binding protein polyacrylamide gel electrophoresis electrophoretic mobility shift assay 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. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar, 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar, 3Landy A. Curr. Opin. Genet. Dev. 1993; 3: 699-707Crossref PubMed Scopus (85) Google Scholar, 4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). Site-specific recombination, mediated by the recombinases of the λ-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 (λ-integrase), of the recombinase. In the second transesterification reaction, the phosphodiester linkages of the exchanged DNA strands are restored (reviewed in Refs. 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar and 3Landy A. Curr. Opin. Genet. Dev. 1993; 3: 699-707Crossref PubMed Scopus (85) Google Scholar). 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 3′-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 3′ hydroxyl groups at the element ends (reviewed in Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar and 4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). These features of the recombination reactions mediated by site-specific recombinases and transposases suggest that a group of related recombinases would not mediate both site-specific recombination and transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DNA inversion inMoraxella lacunata and Moraxella bovis, exhibits significant homology to the transposases of the IS110/IS492 family of IS elements (approximately 25–35% amino acid identity and 45–55% similarity, 5). Furthermore, Piv and the IS110/IS492 transposases do not appear to be related to the site-specific recombinases of the λ-Int or Hin/resolvase families or the transposases containing the DDE motif (4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). Therefore, Piv and the IS110/IS492transposases may define a new family of DNA recombinases. The homology between Piv and the IS110/IS492transposases includes several highly conserved amino acid regions (5Lenich A.G. Glasgow A.C. J. Bacteriol. 1994; 176: 4160-4164Crossref PubMed Google Scholar), which, based on mutational analyses, contain functionally relevant amino acid motifs. 1D. Tobiason, A. Lenich, and A. C. Glasgow, submitted for publication. Although there is no completely conserved DNA sequence among all the IS elements and the Piv invertible DNA segment, there is a consensus sequence for the ends of a subgroup of the IS elements, which is also found overlapping the recombination sites of the Piv invertible element.1 To determine if indeed Piv and the IS110/IS492 transposases define a new family of DNA recombinases that utilize a common mechanism for both site-specific recombination and transposition, we must characterize the recombination reactions mediated by Piv and transposases from the IS110/IS492 family. As a first step in understanding the mechanism for Piv-mediated assembly of a synaptic nucleoprotein complex, DNA cleavage, and strand exchange, we have characterized the interactions of Piv with the invertible DNA segment of Moraxella lacunata. DNase I was obtained from Worthington Biochemical Corp. Amylose resin, restriction enzymes, T4 DNA ligase, Klenow (exo−), and T4 polynucleotide kinase were purchased from New England Biolabs (NEB). Pfu DNA polymerase was obtained from Stratagene. The His tag XPRESS purification system and the TA cloning kit were purchased from Invitrogen. Radionucleotides were obtained from NEN Life Science Products. Isopropyl-1-β-d-galactopyranoside (IPTG),2 dithiothreitol, and EDTA were purchased from Sigma. Dimethyl sulfate was purchased from Aldrich. The pAG607 plasmid is composed of the pMal-C2 vector (NEB) containing the piv gene inserted into theXmnI and BamHI sites such that it is in frame directly downstream of a factor Xa protease site in themalE gene. The piv gene was obtained by the polymerase chain reaction (PCR) from the pMxL1 plasmid, subclone ofM. lacunata inversion region provided by C. Marrs (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar), usingPfu DNA polymerase and the primers: 5′-GCCAGCACGTGTCTAAAACTTACATTG-3′ and 5′-CCTAAGCTTCTAGGATACCAATAAAT-3′. Similarly, the piv gene from pMxL1 was PCR-amplified with the primers: 5′-CTCGTCTCGAGTTCATGAATGCGTTTGTCAAAAG-3′ and 5′-CAGTTCACATATGTCTAAAACTTACATTGGGATT-3′, cleaved withXhoI and NdeI, and inserted into the same restriction sites in pET21a (Novagen). This placed piv in frame with a COOH-terminal His6 tag sequence, creating the Piv-His6 expression vector, pAG1300. A 245-bp fragment was generated from the pMxL1-dl24 plasmid (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar) usingPfu DNA polymerase and the primers 5′-CATAGGATCCAAAATTACCTGCCAGACATC-3′ and 5′-CCGGAATTCGCTAACCTTACACTCATAC-3′. This 245-bp fragment, containing the strong upstream binding site (sub1), was cloned into the pCR2.1 vector (TA cloning kit, Invitrogen). The resulting plasmids, pAG604 and pAG605, contain sub1 in opposite orientations. The inserted DNA was sequenced (T7 Sequenase version 2.0 DNA sequencing kit; Amersham Pharmacia Biotech) to check for any mutations due to the PCR reaction. To construct the DNA inversion test plasmid pAG862, a 5865-bp DNA fragment containing the invertible segment was obtained by partial digestion of pMxL1 with EcoRI. This fragment was gel-purified and ligated into EcoRI-digested pACYC184 (NEB) creating the pAG850 plasmid. To inactivate piv, the Ω fragment encoding the SpcR/SmR genes was recombined into the piv gene of pAG850 by homologous recombination with piv::Ω on the pMxL5 plasmid (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar) in the Rec+ strain JM101 (NEB). The recombinant plasmid, pAG862, was confirmed by restriction digestion and DNA sequencing. To produce the Piv protein fused at its amino terminus to maltose-binding protein (MBP-Piv), DH5α containing pAG607 was grown in Luria broth to OD600 = 0.5, induced with 0.5 at for and in a was an with dithiothreitol, and with in C. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). were and in or at was the protein assay To produce the Piv, was into grown to in Luria induced with at were by in and in a at The was at The was an with protein was with an step and Piv-His6 at The protein was into and at and at to the Piv protein was in with protein were from the by to of DH5α by multiple a bound coli and This was by a of that been induced with 0.5 to the binding the from this to the of the Pharmacia The was at gel electrophoresis was the of Scholar). was the of Scholar) with the was for and the was with a of Piv The protein were low or from DH5α containing pAG862, and pAG607 or containing and pMal-C2 were grown to OD600 = induced with and for at were an Scholar) and with and The restriction were on a The DNA was by and by of the DNA and were with T4 polynucleotide kinase the of Scholar). pAG604 and were with to containing site to be in DNase I protection These were at the site with Klenow DNA polymerase by the with the of the reactions at the were on a polyacrylamide from the and at The plasmid, a of provided by C. Marrs (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar), and the fragment by PCR from usingPfu DNA polymerase and the primers: and were with restriction as The DNA were with or Klenow and at Gel electrophoresis or gel were by protein with DNA fragment in binding dithiothreitol, and (NEB). The reactions were for at a polyacrylamide and at in the competition assays, of DNA from to was to the reaction of the The DNA were the and and the DNA was a to the DNase I protection at the sub1 the from pAG604 and pAG605, on the strand and strand for sub1 were for at with or in binding DNase I protection were in binding with of DNase I as A.C. J. Biol. PubMed Google Scholar). cleavage the and sequencing reactions and were with the same PubMed Scopus Google Scholar). The and sequencing reactions were on a sequencing polyacrylamide the site of sulfate were as J. 1990; PubMed Scopus Google Scholar). DNase I and protection were also at the site of inversion the by and PubMed Scopus Google Scholar) with the for the DNase I protection gel were with DNase I in binding for the reaction with the of Gel electrophoresis were with DNA and of protein from to for binding to and to for binding to of to DNA binding site is by the = which = is the of is the of active is the of active in the is the of bound and is the of A of a with J. Mol. Biol. PubMed Scopus Google Scholar). The of bound and DNA was a with were in binding was with protein as for at was at the were a polyacrylamide gel at the were and with a was to the in the to and The of the DNA in the reaction that was bound by was by the of DNA in the by the DNA in the reaction the and DNA for The bound DNA was as a of the of The for was the for a in due to dissociation of the DNA on the sequence of the piv the Piv protein is to be a acid with of and an of To purification of Piv, M. lacunata piv was into the expression vector the of piv to produce the was and at high was to the protein for in DNA binding A shows a gel containing from a for purification of and from the of protein bound to the of a of a gel containing of the protein that the in all the are protein not shows of expression of the protein from pAG607 and to as be in the of a from DH5α containing pAG607 with 0.5 and The are for Piv as no with was of with the protease factor which a cleavage site between and Piv in the in cleavage at the sequence also within Piv this cleavage site of the Piv protein from by Xa we the protein the of type in inversion assay was to recombinase activity as an of interactions DH5α was with a DNA inversion test plasmid, pAG862, and expression encoding or expression of or recombination of the invertible segment on was by restriction digestion of plasmid In the of the test plasmid, digestion with and in two of and there is the same digestion two different of and no inversion are in the of of the test plasmid is in the In a inversion type Piv also inversion to of an in the inversion reaction not that the is a the protein was in in DNA binding on the binding of site-specific recombinases and transposases (reviewed in Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar and 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar), we that Piv would DNA encoding the cross-over sites for inversion and as as sites in recombination or assay for Piv DNA binding sites within the inversion region was gel electrophoresis mobility shift not with a of restriction that both the invertible segment and the for inversion in coli of the were in the of in and were for by DNase I The fragment was it the left recombination site of the invertible element The fragment also contains the recombination a site DNase I protection with and the fragment and from to upstream of the cross-over site not due to the of the fragment, the DNase I cleavage at the site was not Therefore, a fragment of containing the DNA sequence, was by DNase I protection and The DNase I cleavage in the binding region of no cleavage due to that the DNase I binding and cleavage PubMed Scopus Google Scholar). These are also the DNA to DNA to this region of the DNA and from to and from to and be in DNase I protection regions were on the This binding site is from the site of DNA cleavage and strand in the inversion reaction to be the binding site for the Piv that the recombination reaction. it is that this Piv binding site is an site in the assembly of an active synaptic for Piv-mediated DNA inversion The fragment contains the site of inversion that was based sequence homology at the ends of the invertible segment Marrs C.F. Stevens S.P. J. Bacteriol. 1990; 172: PubMed Google Scholar). The of binding to the fragment that to a sequence within this fragment not DNase I and protection with the fragment and no of interactions not shift the fragment in DNase I protection were with the fragment in which the and DNA are with DNase I in the polyacrylamide gel and from gel for electrophoresis on the DNA sequencing polyacrylamide gel and this assay have for DNA with no protection of DNA sequence by was These that the binding of to the fragment was or not To determine if the interactions of with due to the protein at the amino terminus of Piv, Piv was at the terminus with and The Piv-His6 protein was and in and DNase I protection with the fragment and also with the fragment containing the Piv binding site not of the DNA binding with Piv-His6 with obtained with that the binding of to not due to by of Therefore, binding of Piv may such as DNA binding DNA to binding to the recombination sites To characterize the interactions of with the and upstream to site were and The sequence of the region of homology from of the invertible element and of sequence upstream and downstream from the The DNA sequence of upstream binding site was based on the DNase I obtained with the DNA fragment the region to not the to The and DNA binding were in an to binding at site and were with the that at two the not the DNA sequence from to we have the binding site within the binding to in that at the same rate as the with the in the of the and is in to the this result that the and contain the same of was with the of was not The of a was with in the The formation of this that a may or an may the the of and the electrophoretic of and indicate that is bound by at two protomers is bound by or multiple of the Piv DNA binding and homology that Piv two different DNA recognition To the interactions of with the different binding sites on and competition were of the as both binding and In the was and in DNA binding with and the and the and Although for both and formation at and for formation at the same of This result is with with in a The DNA also with for binding to This competition for formation by the may indicate that protein a rate and it is of the reaction by interactions at shows that the for binding to not significant competition at These suggest a different interaction between as with binding of The of the DNase I gel shift assays, and DNA binding competition be as that the interactions of with are with The to of the DNA bound with of be by a dissociation rate for bound to the site of such that no of the with be by the DNA binding rate with of that the bound DNA in the of with the DNA any formation there was to a of the with The dissociation rate with a for the of to the The complex, a of that binding as a in this is The apparent dissociation rate of and is also with of an protein interactions binding of multiple to DNA Therefore, binding of may formation with the or sites suggest that multiple protomers of are a of may if in binding to the To determine was a is the of protein and and are the of the bound and if binds as a to the to that the binding of the two is to that the binding is not and the is binding to that DNA site J. Mol. Biol. PubMed Scopus Google Scholar). To determine the cooperativity of binding in the and of were with the DNA fragment encoding the site and by on the that the complex, the of bound to DNA was and with the of The the was for binding reactions with to The of the resulting is binding by in the of the that Piv is binding The the was for binding reactions with to and the resulting a of that the binding is This result the that at two are binding to the in the was also for Piv binding to the site of inversion the The with of no intermediate was The the was for and the of the resulting is this result that binding is Therefore, must assemble in or at site binding to The site-specific recombinase Piv is a of the novel family of DNA recombinases that includes the IS110/IS492transposases (5Lenich A.G. Glasgow A.C. J. Bacteriol. 1994; 176: 4160-4164Crossref PubMed Google Scholar). To determine the mechanism for DNA cleavage and strand mediated by this DNA we must how Piv interacts with the DNA segment that it in M. lacunata. have DNA binding studies with and DNA from the type IV pilin inversion region of M. lacunata. The activity of the protein in gel electrophoresis DNA binding and nuclease/chemical protection is to to the activity of type Piv, as is active in for DNA inversion of the DNA sequence of the inversion region ofM. lacunata which the left and ends of the invertible element Marrs C.F. Stevens S.P. J. Bacteriol. 1990; 172: PubMed Google Scholar, Marrs C. J. Bacteriol. PubMed Google Scholar). Piv-mediated DNA cleavage and strand must within two for site-specific DNA inversion of the pilin Therefore, based on site-specific recombination and transposition systems N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar, 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar), it that Piv binds to a DNA sequence overlapping or and The of electrophoretic mobility shift indicate that indeed with DNA containing both and as as with a DNA from both outside and within the invertible DNA segment of M. lacunata The binding of to and of the sites located outside of the invertible segment was interactions with the site are as by the gel shift DNA binding in which of the DNA or DNA be no how high the of protein and not In not a at site in or chemical protection of the dissociation rate for the bound or the fragment, not that the is the of the is this binding of to have been due to the of the maltose-binding protein at the amino terminus of Piv, we found that Piv containing a His6 tag at the terminus also bound Piv is not the first site-specific recombinase to for the site it DNA cleavage and strand exchange. λ binds to the sites it DNA cleavage and strand at or interactions with Int bound to the sites and DNA mediated by binding of Int to the sites and A. J. Mol. Biol. PubMed Scopus Google Scholar, A. 1990; PubMed Scopus Google Scholar). Int binds with low to the sites A. PubMed Scopus Google Int not a DNase I at the site of in the of the and sites A. J. Mol. Biol. PubMed Scopus Google Scholar). λ Int is a two DNA binding The Int binds with high to the the binds to the sites and DNA cleavage and strand exchange. of Int the and sites within and sites in A. 1990; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Piv binds to the we not it surprising Piv binds DNA that may the formation of a synaptic The protection and gel shift DNA binding that Piv binds with to a different DNA recognition sequence located upstream of and and A and This strong upstream binding site is bound by at two protomers of The dissociation rate with the site suggest that the first that binds to a the is The interactions of with appear to from with not in the and also in the of the protein for binding to the The cooperativity and competition suggest that the of binds to protomers of to this is not for a protein that DNA The transposase of binds as a to sites within the sequence which assembly of a synaptic structure PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). the recombination sites at the ends of the transposable element with a different of the and a that is bound to both ends of the element on a DNA PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The interactions of with the recombination sites and it is this that is active for recombination Dev. PubMed Scopus Google Scholar). In reaction a binding a recombination not on the sites Dev. PubMed Scopus Google Scholar). Therefore, we the of the and it is that site is an assembly site for the of Piv that binds to the recombination The of the with may the for within a synaptic complex, the binding of λ to the Piv interacts with the sites within an structure and the of the recombination reaction. As been for a of transposition and site-specific recombination systems Dev. 1998; PubMed Scopus Google for Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar and 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar), it is for the recombinase to be active at recombination sites a synaptic structure is this that the strand and DNA as an assembly site for Piv, it is to the rate of inversion or to be for Piv-mediated DNA of the M. segment and DNA have that the region encoding the site is not essential for inversion of the Moraxella DNA segment in it may have an on the rate of inversion J. Mol. Biol. PubMed Scopus Google Scholar). the site in λ PubMed Scopus Google Scholar), the site may be such as recombinase are low or in the of of the sequence of the inversion region the with the site sequence two sites within the and DNA that were bound by in the gel shift DNA binding not These sites may for the site or the inversion reaction with In to define the of Piv and its different binding sites in the inversion reaction, we are in recombination to in the DNA inversion reaction. and for of this and are to for to the of