An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage

Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.     

R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide

F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.     

Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.Transfer of conjugative plasmids between bacteria contributes to genome diversification and acquisition of new traits. Conjugative plasmids encode most proteins required for transfer of one plasmid strand from the donor to the recipient cell (reviewed in references 11, 24, and 43). In preparation for transfer, a complex of proteins assembles at the plasmid origin of transfer (oriT). Within this complex, called the relaxosome, a plasmid-encoded relaxase or nickase binds and cleaves one plasmid strand at a specific oriT site (nic). As part of the cleavage reaction, the relaxase forms a covalent linkage between an active-site tyrosyl hydroxyl oxygen and a single-stranded DNA (ssDNA) phosphate, yielding a 3′ ssDNA hydroxyl (19, 30). Upon initiation of transfer, the plasmid strands are separated, and the cut strand is transported into the recipient. The relaxase is likely transferred into the recipient (12, 31) while still physically attached to plasmid DNA. The transferred relaxase may then join the ends of the ssDNA plasmid copy in the final step of plasmid transfer. Complementary strand synthesis in the donor and the recipient generates a double-stranded plasmid that is competent for further transfer. Successful conjugation requires effective temporal regulation, yet the mechanisms governing this regulation are poorly understood.The F plasmid oriT is ∼500 bp long and includes multiple binding sites for integration host factor (IHF), TraY, and TraM and a single site for TraI, the F relaxase (11). IHF, TraY, and TraM, participants in the relaxosome, bind double-stranded DNA to facilitate the action of TraI, perhaps by creating or stabilizing the ssDNA conformation around nic required for TraI recognition. The F TraI minimal high-affinity binding site includes ∼15 nucleotides around nic (39), and throughout the text, we refer to oligonucleotides that contain the TraI wild-type (wt) or variant binding site as oriT oligonucleotides. F TraI is 192 kDa (42), and in addition to its relaxase activity, TraI has a 5′-to-3′ helicase activity (4). These activities must be physically joined to allow efficient plasmid transfer (29), yet how the two activities are coordinated is a mystery. The relaxase region of F TraI has been defined as the N-terminal ∼300 amino acids (aa) (6, 40). Conserved helicase motifs, including those associated with an ATPase, lie between amino acids 990 and 1450. The C-terminal region (positions 1450 to 1756) plays an important role in bacterial conjugation, possibly involving protein-protein interactions with TraM (32) and/or inner membrane protein TraD (28).The 70-kDa central region of TraI that lies between the relaxase and helicase domains has been implicated in two functions. Haft and colleagues described TraI variants with 31-amino-acid insertions in this TraI region that facilitated plasmid transfer with greater efficiency than that afforded by the wild-type protein when these proteins are expressed at high levels (16). On the basis of this observation, the authors proposed that the region participated in a negative regulation of transfer. Matson and Ragonese demonstrated that this central region is required for TraI helicase function, likely due to participation in ssDNA recognition essential for the helicase activity (28). We wondered whether the proposed regulatory and ssDNA binding roles of the central region are linked and whether this region might help modulate TraI helicase and relaxase activities. Our objectives in this study were to confirm the role of the central region in ssDNA recognition, to assess the affinity and specificity of the ssDNA recognition by the central region, and to determine whether the relaxase and central domain ssDNA binding sites demonstrate cooperativity in binding. Our work yielded two significant and surprising results. First, the binding site within the TraI central region binds ssDNA with high affinity and significant sequence specificity, both unusual characteristics for a helicase. Second, the central region and relaxase ssDNA binding sites show an apparent strong negative cooperativity of binding, possibly explaining the role of the central region as a negative regulator and providing clues about how the timing of conjugative transfer might be regulated.     

DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA

The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.     

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.     

Energetics of the sequence-specific binding of single-stranded DNA by the F factor relaxase domain

Transfer of conjugative plasmids between bacteria requires the activity of relaxases or mobilization proteins. These proteins nick the plasmid in a site- and strand-specific manner prior to transfer of the cut strand from donor to recipient. TraI36, the relaxase domain of TraI from plasmid F factor, binds a single-stranded DNA (ssDNA) oligonucleotide containing an F factor sequence with high affinity and sequence specificity. To better understand the energetics of this interaction, we examined the temperature, salt, and pH dependence of TraI36 recognition. Binding is entropically driven below 25 degrees C and enthalpically driven at higher temperatures. van't Hoff analysis yields an estimated deltaC(P)(0) of binding (-3300 cal x mol(-1) x K(-1)) that is larger and more negative than that observed for most double-stranded DNA (dsDNA)-binding proteins. Based on analyses of circular dichroism data and the crystal structure of the unliganded protein, we attribute the deltaC(P)(0) to both burial of hydrophobic surface area and coupled folding and binding of the protein. The salt dependence of the binding indicates that several ssDNA phosphates are buried in the complex, and the pH dependence of the binding suggests that some of these ssDNA phosphates form ionic interactions with basic residues of the protein. Although data are available for relatively few sequence-specific ssDNA-binding proteins, sufficient differences exist between TraI36 and other proteins to indicate that, like dsDNA-binding proteins, ssDNA-binding proteins use different motifs and combinations of forces to achieve specific recognition.     

The relaxase of the Rhizobium etli symbiotic plasmid shows nic site cis-acting preference

Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.     

Structural insights into single-stranded DNA binding and cleavage by F factor TraI

Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.     

Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5'-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3'-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3'-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3'-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.     

General mutagenesis of F plasmid TraI reveals its role in conjugative regulation

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.     

A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation

Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC–DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome.     

Unveiling the molecular mechanism of a conjugative relaxase: The structure of TrwC complexed with a 27-mer DNA comprising the recognition hairpin and the cleavage site

TrwC is a DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein-DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn2+, Ni2+ and Cu2+ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase.     

Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.     

The F plasmid-encoded TraM protein stimulates relaxosome-mediated cleavage at oriT through an interaction with TraI

Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer - they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5'-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.     

TraY and integration host factor oriT binding sites and F conjugal transfer: sequence variations, but not altered spacing, are tolerated

Bacterial conjugation is the process by which a single strand of a conjugative plasmid is transferred from donor to recipient. For F plasmid, TraI, a relaxase or nickase, binds a single plasmid DNA strand at its specific origin of transfer (oriT) binding site, sbi, and cleaves at a site called nic. In vitro studies suggest TraI is recruited to sbi by its accessory proteins, TraY and integration host factor (IHF). TraY and IHF bind conserved oriT sites sbyA and ihfA, respectively, and bend DNA. The resulting conformational changes may propagate to nic, generating the single-stranded region that TraI can bind. Previous deletion studies performed by others showed transfer efficiency of a plasmid containing F oriT decreased progressively as increasingly longer segments, ultimately containing both sbyA and ihfA, were deleted. Here we describe our efforts to more precisely define the role of sbyA and ihfA by examining the effects of multiple base substitutions at sbyA and ihfA on binding and plasmid mobilization. While we observed significant decreases in in vitro DNA-binding affinities, we saw little effect on plasmid mobilization even when sbyA and ihfA variants were combined. In contrast, when half or full helical turns were inserted between the relaxosome protein-binding sites, mobilization was dramatically reduced, in some cases below the detectable limit of the assay. These results are consistent with TraY and IHF recognizing sbyA and ihfA with limited sequence specificity and with relaxosome proteins requiring proper spacing and orientation with respect to each other.     

Mechanistic Basis of Plasmid-Specific DNA Binding of the F Plasmid Regulatory Protein,TraM

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.     

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 Å crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 Å out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid’s origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.     

Location of the nick at oriT of the F plasmid

The oriT locus of the Escherichia coli K12 F plasmid contains a site at which one of the DNA strands is cleaved as a prelude to conjugal transmission to recipient bacteria. We have remapped this site biochemically by using oriT-containing plasmids that were purified from bacteria expressing the F transfer (tra) functions. The strand interruption was found on the transferred strand 137 base-pairs clockwise of the center of the BglII site at 66.7 on the F map. This location is consistent with the locations anticipated from studies of delta traF' plasmids, but it differs from previous results by other investigators. The strand interruption produced a 3'-OH, but the nature of the 5' terminus of the strand on the other side of the nick was not determined. Some DNA sequence motifs in the vicinity of the oriT nick site of F resemble the chromosomal site involved in formation of delta traF'purE plasmids.     

Roles of active site residues and the HUH motif of the F plasmid TraI relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.