Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

Nicking by transesterification: the reaction catalysed by a relaxase

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site- and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3'-OH and the relaxase covalently bound to the 5' end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.     

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.     

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.     

F plasmid conjugative DNA transfer: the TraI helicase activity is essential for DNA strand transfer

The product of the Escherichia coli F plasmid traI gene is required for DNA transfer via bacterial conjugation. This bifunctional protein catalyzes the unwinding of duplex DNA and is a sequence-specific DNA transesterase. The latter activity provides the site- and strand-specific nick required to initiate DNA transfer. To address the role of the TraI helicase activity in conjugative DNA transfer traI mutants were constructed and their function in DNA transfer was evaluated using genetic and biochemical methods. A traI deletion/insertion mutant was transfer-defective as expected. A traI C-terminal deletion that removed the helicase-associated motifs was also transfer-defective despite the fact that the region of traI encoding the transesterase activity was intact. Biochemical studies demonstrated that the N-terminal domain was sufficient to catalyze oriT-dependent transesterase activity. Thus, a functional transesterase was not sufficient to support DNA transfer. Finally, a point mutant, TraI-K998M, that lacked detectable helicase activity was characterized. This protein catalyzed oriT-dependent transesterase activity in vitro and in vivo but failed to complement a traI deletion strain in conjugative DNA transfer assays. Thus, both the transesterase and helicase activities of TraI are essential for DNA strand transfer.     

The R1162 relaxase/primase contains two, type IV transport signals that require the small plasmid protein MobB

The relaxase of the plasmid R1162 is a large protein essential for conjugative transfer and containing two different and physically separate catalytic activities. The N-terminal half cleaves one of the DNA strands at the origin of transfer (oriT) and becomes covalently linked to the 5' terminal phosphate; the C-terminal half is a primase essential for initiation of plasmid vegetative replication. We show here that the two parts of the protein are independently transported by the type IV pathway. Part of the domain containing the catalytic activity, as well as an adjacent region, is required in each case, but the required regions do not physically overlap. Both transport systems contribute to the overall frequency of conjugative transfer. MobB is a small protein, encoded within mobA but in a different reading frame, that stabilizes the relaxase at oriT. MobB is required for efficient type IV transport of both the complete relaxase and its two, separate functional halves. MobB inserts into the membrane and could thus stabilize the association between the relaxase and the type IV transfer apparatus.     

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.     

Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation

The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT-mobB-mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5'-GGGTG/GTCGGG-3' by primer extension and sequencing reactions. Analysis of Mob- insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which nic cleavage results in a free nicked-DNA intermediate.     

Subdomain organization and catalytic residues of the F factor TraI relaxase domain

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.     

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.     

Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a beta-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.     

The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.     

Solution structure and small angle scattering analysis of TraI (381-569)

TraI, the F plasmid-encoded nickase, is a 1756 amino acid protein essential for conjugative transfer of plasmid DNA from one bacterium to another. Although crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. The central region (between residues 306 and 1520) is known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Here, we show that the ssDNA binding site is located between residues 381 and 858, and we also present the high-resolution solution structure of the N-terminus of this region (residues 381-569). This fragment folds into a four-strand parallel β sheet surrounded by α helices, and it resembles the structure of the N-terminus of helicases such as RecD and RecQ despite little sequence similarity. The structure supports the model that F TraI resulted from duplication of a RecD-like domain and subsequent specialization of domains into the more N-terminal ssDNA binding domain and the more C-terminal domain containing helicase motifs. In addition, we provide evidence that the nickase and ssDNA binding domains of TraI are held close together by an 80-residue linker sequence that connects the two domains. These results suggest a possible physical explanation for the apparent negative cooperativity between the nickase and ssDNA binding domain.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.

Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.     

Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.     

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.     

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.     

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Background

Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/Principal Findings

We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/Significance

The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.     

Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases

In preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5' end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB(F) and MOB(Q) families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.