Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.     

Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata

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 lambda 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.     

Diversity in the serine recombinases

Most site-specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or lambda integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N-terminal catalytic domain and a C-terminal DNA binding domain. Phage-encoded serine recombinases (e.g. phiC31 integrase) cause integration and excision with strictly controlled directionality, and have an N-terminal catalytic domain but much longer C-terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N-terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.     

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.     

Retroviral integrase superfamily: the structural perspective

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid‐processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA‐mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus—RNase H1 and integrase—are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

Presence of a characteristic D-D-E motif in IS1 transposase

Transposases encoded by various transposable DNA elements and retroviral integrases belong to a family of proteins with three conserved acidic amino acids, D, D, and E, constituting the D-D-E motif that represents the active center of the proteins. IS1, one of the smallest transposable elements in bacteria, encodes a transposase which has been thought not to belong to the family of proteins with the D-D-E motif. In this study, we found several IS1 family elements that were widely distributed not only in eubacteria but also in archaebacteria. The alignment of the transposase amino acid sequences from these IS1 family elements showed that out of 14 acidic amino acids present in IS1 transposase, three (D, D, and E) were conserved in corresponding positions in the transposases encoded by all the elements. Comparison of the IS1 transposase with other proteins with the D-D-E motif revealed that the polypeptide segments surrounding each of the three acidic amino acids were similar. Furthermore, the deduced secondary structures of the transposases encoded by IS1 family elements were similar to one another and to those of proteins with the D-D-E motif. These results strongly suggest that IS1 transposase has the D-D-E motif and thus belongs to the family of proteins with the D-D-E motif. In fact, mutant IS1 transposases with an amino acid substitution for each of the three acidic amino acids possibly constituting the D-D-E motif were not able to promote transposition of IS1, supporting this hypothesis. The D-D-E motif identified in IS1 transposase differs from those in the other proteins in that the polypeptide segment between the second D and third E in IS1 transposase is the shortest, 24 amino acids in length. Because of this difference, the presence of the D-D-E motif in IS1 transposase has not been discovered for some time.     

Mechanism of action of RNase T. I. Identification of residues required for catalysis,substrate binding,and dimerization

Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.     

Site-specific recombinases as tools for heterologous gene integration

Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.     

Amino acid sequence homology between Piv, an essential protein in site-specific DNA inversion in Moraxella lacunata, and transposases of an unusual family of insertion elements.

Deletion analysis of the subcloned DNA inversion region of Moraxella lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tfpQ/tfpI pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase.     

Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination.

The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.     

Comparative architecture of transposase and integrase complexes

Transposases and retroviral integrases promote the movement of DNA segments to new locations within and between genomes. These recombinases function as multimeric protein-DNA complexes. Recent success in solving the crystal structure of a Tn5 transposase--DNA complex provides the first detailed structural information about a member of the transposase/integrase superfamily in its active, DNA-bound state. Here, we summarize the reactions catalyzed by transposases and integrases and review the Tn5 transposase-DNA co-crystal structure. The insights gained from the Tn5 structure and other available structures are considered together with biochemical and genetic data to discuss features that are likely to prove common to the catalytic complexes used by members of this important protein family.     

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.     

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn²⁺ binds tightly to the sulfur atoms of the cysteine residues, but Mg²⁺ does not. We found that Mn²⁺, but not Mg²⁺, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn²⁺ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.     

Comparative architecture of transposase and integrase complexes.

Transposases and retroviral integrases promote the movement of DNA segments to new locations within and between genomes. These recombinases function as multimeric protein-DNA complexes. Recent success in solving the crystal structure of a Tn5 transposase--DNA complex provides the first detailed structural information about a member of the transposase/integrase superfamily in its active, DNA-bound state. Here, we summarize the reactions catalyzed by transposases and integrases and review the Tn5 transposase-DNA co-crystal structure. The insights gained from the Tn5 structure and other available structures are considered together with biochemical and genetic data to discuss features that are likely to prove common to the catalytic complexes used by members of this important protein family.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.