The traY gene product and integration host factor stimulate Escherichia coli DNA helicase I-catalyzed nicking at the F plasmid oriT.

F plasmid conjugative transfer is initiated by the introduction of a site- and strand-specific nick within the plasmid origin of transfer (oriT). Genetic studies have shown nick formation to be dependent on both the traI and traY genes. However, highly purified TraIp, the traI gene product, nicks oriT in a site- and strand-specific manner in the absence of the traY gene product (TraYp) in vitro (Matson, S.W., and Morton, B.S. (1991) J. Biol. Chem. 266, 16232-16237). Analysis of the oriT region has revealed binding sites for TraYp and the host protein integration host factor (IHF). To explore possible interactions occurring at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate. A marked enhancement of the nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF. In addition, TraIp was able to nick a linear oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone. Individual protein concentration requirements for the supercoiled and linear nicking reactions were similar, and the reactions occurred at equal velocity, suggesting that they are biochemically identical. Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported KD values for the IHF and TraYp binding sites in oriT. These data, coupled with data presented in the accompanying report, suggest that TraYp and IHF bind independent of one another, forming a nucleo-protein complex with oriT that can be recognized and nicked by TraIp.

The F plasmid is a 100-kilobase pair self-transmissible plasmid that inhabits many Escherichia coli strains. During a mating event, a single strand of the F plasmid is transferred, with the 5Ј-end leading, from the donor bacterium to the recipient bacterium. The transferred genetic material is stabilized in the recipient either by recombination into the chromosome or complementary strand synthesis (for reviews, see Refs. [1][2][3]. Myriad other transmissible plasmids have been described that inhabit various species of bacteria, allowing this type of horizontal gene transfer to occur in an intra-or extraspecies-specific manner. The tra region (for transfer) on the F plasmid encodes essentially all of the plasmid genes necessary to support bacterial conjugation (2). Of the 36 known genes encoded in this region, only four, traM, traY, traD, and traI, have been shown to be directly involved in DNA mobilization (1). Two of these genes, traY and traI, are required for formation of a site-and strandspecific nick at the origin of transfer (oriT) (4). The formation of the site-and strand-specific nick in oriT is generally considered the first step in DNA mobilization. TraMp and TraDp have been shown not to be involved in nick formation in vivo and are proposed to play a role in subsequent steps of mobilization. As such, they were not considered in this study.
The traI gene encodes DNA helicase I (5), an enzyme that has been well characterized in terms of both its DNA unwinding activity and its DNA-dependent ATPase activity (6 -9). More recently, we and others have shown that TraIp also contains the catalytic site responsible for site-and strandspecific cleavage at oriT (10,11). The large size of TraIp, in comparison with other known bacterial helicases, suggests the possibility of separate nicking and helicase domains. This notion is further supported by mutational analysis that localized nicking activity to the amino-terminal half of the protein and helicase activity in the carboxyl-terminal half (5,9,12). The oriT-specific nicking reaction catalyzed by TraIp requires magnesium and an oriT-containing DNA substrate that is either supercoiled or single-stranded (10,11,13). Cleavage occurs at exactly the same phosphodiester bond that is nicked in vivo. As the phosphodiester bond is cleaved, a covalent linkage forms between TraIp and the 5-phosphate of the nicked strand. Thus bond scission is the result of transesterification and not hydrolysis (14). The nicked product observed is actually a stable reaction intermediate. Consistent with the notion of a reversible transesterification, TraIp has been shown to reseal the break formed in the phosphodiester backbone (13). TraIp, therefore, likely plays a role in both the initiation and termination of strand transfer.
The product of the traY gene (TraYp) is a site-specific DNAbinding protein with three known binding sites (15)(16)(17). Two of these binding sites are located within oriT within 100 bp 1 of nic, the site that is nicked in vivo to initiate strand transfer. The position of these sites suggests the possibility of proteinprotein contacts between TraIp and TraYp during the initiation of strand transfer. However, no such interactions have been demonstrated, and the addition of TraYp has no impact on the oriT-specific nicking reaction catalyzed by TraIp (10). The third TraYp binding site is coincident with the mRNA start site of the traYI operon and is proposed to be involved in transcriptional regulation (15).
Integration host factor (IHF) binds two specific sites within oriT (18). IHF is a heterodimer encoded by the chromosomal genes himA and hip (19). It is involved in a wide variety of cellular processes including replication, transcription, and recombination. Moreover, IHF has been shown to play a role in expression of F tra genes. The identification of binding sites in oriT was the first indication that IHF might play a role in DNA metabolism during conjugative transfer (20).
Genetic studies have suggested that TraYp is involved in nick formation in vivo, and it is known that the protein binds oriT near the nic locus. Therefore, a role for TraYp in the in vitro reaction catalyzed by TraIp, which has been elusive, might be uncovered by exploring the roles of other proteins known to bind oriT. To this end, combinations of IHF, TraYp, and TraIp were incubated with supercoiled and linear oriTcontaining DNA substrates. TraYp and IHF together stimulate the TraIp-catalyzed reaction on a supercoiled DNA substrate and confer recognition of a linear DNA as a substrate.

Materials
Enzymes-DNA polymerase I large fragment, T4 polynucleotide kinase, and restriction enzymes were purchased from Amersham Corp., New England Biolabs, or Boehringer Mannheim and used according to the supplier's specifications.
TraIp was purified using a modification of the protocol described previously (21). Fractions I-III were prepared as described previously (21). Fraction III was dialyzed against buffer A (50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 20% glycerol) containing 100 mM NaCl, and loaded onto a 10-ml (1.5 cm ϫ 5.5 cm) heparinagarose (Sigma) column equilibrated with buffer A containing 50 mM NaCl. The column was washed with 5 column volumes of buffer A containing 200 mM NaCl and eluted using a 10-column volume linear gradient from 200 to 800 mM NaCl in buffer A. Fractions were assayed for single-stranded DNA-dependent ATPase activity (21), which eluted at 400 mM NaCl. Active fractions were pooled and concentrated to 4.5 ml using a Centriprep 30 concentrator (Centricon) (Fraction IV). An 80 ml (1.5 cm ϫ 48 cm) S-200 Superfine (Pharmacia Biotech Inc.) column was equilibrated with buffer A containing 500 mM NaCl, and Fraction IV was applied. Active fractions eluting in the void volume were pooled (Fraction V). Fraction V was adjusted to a final concentration of 50% glycerol and stored at Ϫ70°C. Purified TraIp was greater than 90% homogeneous as judged by electrophoresis in the presence of SDS (Fig. 1A). IHF was purified as described by Nash et al. (22) with the following modifications. Fractions containing IHF, which eluted from the phosphocellulose (Whatman) column, were pooled and adjusted to the conductivity of TG (50 mM Tris-HCl (pH 7.4), 10% glycerol) containing 350 mM KCl by dilution with TG. This pool was loaded on a second phosphocellulose column (0.5 cm ϫ 2.55 cm) equilibrated with TG containing 350 mM KCl. The column was washed with 3 volumes of the same buffer, and protein was eluted using a 20-column volume linear gradient from 350 mM KCl to 1 M KCl in TG. Fractions containing IHF were pooled and dialyzed against a storage buffer containing 50 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 350 mM KCl, 1 mM dithiothreitol, and 50% glycerol. This second phosphocellulose column was found to be necessary to remove contaminants with nuclease activity. The protein was judged to be greater than 95% homogeneous by SDS-PAGE (Fig. 1B).
TraY protein was purified using a modification of the procedure of Nelson et al. (15). Fraction II was dialyzed against buffer A containing 100 mM NaCl and loaded on a 20-ml (2.5 cm ϫ 4.5 cm) heparin-agarose column equilibrated with the same buffer. The column was washed with 2 column volumes of buffer A containing 100 mM NaCl and eluted using a 10-column volume linear gradient from 100 mM to 1 M NaCl in buffer A. Fractions were assayed using a gel mobility shift assay as described previously (15). Active fractions were pooled and dialyzed against buffer A containing 1 M ammonium sulfate (Fraction III). Fraction III was loaded onto a 7-ml (1.5 cm ϫ 4 cm) phenyl-Sepharose (Pharmacia) column equilibrated with buffer A containing 1 M ammonium sulfate. The column was washed with 5 column volumes of the equilibration buffer and eluted using a linear gradient from 1 M to 0 M ammonium sulfate in buffer A. Active fractions, which eluted at 400 mM ammonium sulfate, were pooled and dialyzed against buffer A containing 50 mM NaCl (fraction IV). A 2.5-ml (1 cm ϫ 3 cm) double-stranded DNA cellulose (Amersham Corp.) column was equilibrated with buffer A containing 50 mM NaCl. Fraction IV was applied, the column was washed with 5 column volumes of buffer A containing 100 mM NaCl, and the protein was eluted with a 10-column volume linear gradient from 100 to 800 mM NaCl in buffer A. A gel mobility shift assay and a nuclease assay were performed on fractions from this column. The nuclease assay was identical to the gel mobility shift assay, except the reactions contained 10 mM MgCl 2 and no EDTA. Nuclease activity, indicated by appearance of free labeled nucleotide on the binding gel, was undetectable under the conditions used. The final pool was concentrated using a Centriprep10 concentrator (Centricon), and adjusted to 50% glycerol. The protein was judged to be Ͼ90% homogeneous by SDS-PAGE (Fig. 1B).
The concentration of protein in the purified fraction was determined by the method of Lohman (23). The theoretical extinction coefficient of TraYp was calculated to be 10,870 M Ϫ1 by the PEPTIDESORT program of the GCG software package. Comparing the absorption of equal amounts of TraYp under denaturing and nondenaturing conditions showed the extinction coefficient of the native protein to be equal to the calculated extinction coefficient of TraYp.
The active fraction of TraYp was determined by a modification of the method of Riggs et al. (24). Briefly, gel mobility shift assays were performed (as described above) in which the 32 P-labeled DNA substrate was titrated (17.1-171 nM) against a fixed concentration of TraYp (85 nM). The fraction of substrate that was bound was determined for each reaction by PhosphorImager analysis. A double-reciprocal plot of substrate concentration versus bound complex concentration yields the reciprocal of the concentration of active protein as the y intercept and the negative reciprocal of the K D of TraYp for the substrate as the x intercept. In this manner, the active fraction of this TraYp preparation was determined to be 19.1%. The K D of TraYp for sbyA was 36.6 nM.
DNAs and Nucleotides-The plasmid pBSoriT has been described previously (10). The substrate used in the linear nicking assays was a pBSoriT restriction fragment prepared as follows: pBSoriT DNA was cleaved to completion with XbaI and labeled at the 3Ј-end using [␣-32 P]dCTP and DNA polymerase I large fragment under reaction conditions suggested by the supplier. This linear DNA fragment was then cleaved with SalI and SacII, resulting in the formation of three DNA fragments: a 545-bp 32 P-labeled DNA fragment containing the oriT region, a 13-bp 32 P-labeled DNA fragment, and a 2887-bp unlabeled fragment.
Nucleotides were purchased from Pharmacia-P/L Biochemicals. [␣-32 P]dCTP was obtained from Amersham, Corp. DNA concentrations are expressed in terms of mol of DNA molecules.

Methods
Supercoiled Nicking Assay-Reactions were performed as described previously (10). Briefly, reaction mixtures (20 l) contained 6.7 nM supercoiled pBSoriT DNA, 40 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 , 5 mM dithiothreitol, 15% glycerol, and 25 g/ml bovine serum albumin. Where indicated, poly(dI-dC)⅐poly(dI-dC) (Pharmacia) was present at 12.5 g/ml. Reactions were incubated at 37°C for 30 min, stopped using 4 l of 50 mM EDTA, 0.5% SDS, 250 g/ml proteinase K, and incubated at 37°C for an additional 15 min. Reaction products were resolved on a 0.8% agarose gel run in the absence of ethidium bromide. The gel was stained using ethidium bromide. The fraction of nicked product formed was quantified by irradiating the stained agarose gel with 254-nm light for 30 min to nick all DNA species. This method allowed the nicked and supercoiled species of DNA to intercalate similar amounts of ethidium bromide. The gel was further stained with ethidium bromide, destained in water, and photographed. The photographic negative was scanned using a laser densitometer to quantitate relative amounts of nicked and supercoiled DNA.
Linear Nicking Assay-Reaction conditions were as described for the nicking assay using the supercoiled DNA substrate. The linear DNA substrate was present at ϳ500 pM in each reaction. Reaction products were resolved on a 6% polyacrylamide (20:1 cross-linking), 8 M urea denaturing gel, using 90 mM Tris borate, 2 mM EDTA as a running buffer. Electrophoresis was performed for 2.5 h at 10 watts. Results were quantified using a Molecular Dynamics PhosphorImager.

TraYp and IHF Stimulate the Nicking Reaction Catalyzed by
TraIp-An in vitro reaction has been described previously in which purified TraIp catalyzes a site-and strand-specific transesterification reaction at the nic locus in oriT (10,11). The reaction requires MgCl 2 and a supercoiled DNA substrate containing oriT; relaxed and linear DNA molecules are not substrates for this reaction. Surprisingly, the addition of TraYp, which genetic studies indicate is required for site-and strandspecific nicking in vivo, is not required for the in vitro reaction catalyzed by TraIp (10). Further characterization of this reaction has shown it to be very sensitive to the addition of increasing concentrations of NaCl (Fig. 2) or potassium glutamate (data not shown). The nicking reaction was reduced by more than 70% in the presence of 75 mM NaCl and was essentially undetectable when the NaCl concentration was increased above 100 mM. These properties seem to be inconsistent with the notion that TraIp, by itself, catalyzes the site-and strandspecific nicking reaction that initiates bacterial conjugation in vivo.
Both TraYp and IHF binding sites have been located within the F plasmid oriT (15,17,18). These sites lie near, but not coincident with, the nic locus, and it was reasoned that these two proteins in combination might stimulate the transesterification reaction performed by TraIp. To test this possibility, various combinations of TraYp, IHF, and TraIp were incubated with a plasmid DNA substrate containing oriT in the presence of 75 mM NaCl. The increased concentration of NaCl was included in the reaction to reduce the site-and strand-specific nicking catalyzed by TraIp alone. The results of this experiment are presented in Fig. 3. Incubation of the plasmid DNA with TraIp resulted in the production of a minimal amount of nicked DNA (Fig. 3, lane 4) as expected under these conditions. TraYp and IHF also failed to nick the supercoiled plasmid (Fig.  3, lanes 2 and 3). Moreover, the addition of either TraYp or IHF to reaction mixtures containing TraIp had no effect on the amount of nicked DNA product formed (Fig. 3, lanes 6 and 7). However, when TraYp and IHF were incubated together with TraIp, there was a dramatic increase in the amount of nicked DNA produced (Fig. 3, lane 8). The plasmid was nicked at the same site and on the same strand that is nicked in vivo (data not shown). Quantitation of this data indicated that nicked molecule formation in the three-protein reaction was approximately 7-fold greater than in the reaction catalyzed by TraIp alone. The nicking reaction catalyzed by TraIp in the presence of both TraYp and IHF was also more resistant to increased NaCl concentrations than the reaction catalyzed by TraIp alone (see Fig. 2). Significant nicking of the plasmid DNA was still detectable at NaCl concentrations exceeding 150 mM. Thus, under these conditions, stimulation of the transesterification reaction catalyzed by TraIp is absolutely dependent on the addition of both TraYp and IHF.
To determine the optimal concentrations of TraIp, TraYp, and IHF required for nicking the supercoiled DNA substrate, a series of titrations was performed. In each case, two of the protein components were held at constant concentrations, The fraction of input plasmid that was nicked was quantified as described under "Experimental Procedures." There was a small (Ͻ5%) contamination of nicked DNA in the supercoiled pBSoriT preparation that was subtracted from all data points. The data represents the averages of multiple independent determinations. Error bars represent the standard deviation about the mean.
FIG. 3. TraYp and IHF stimulate the nicking reaction catalyzed by TraIp. Nicking reactions using supercoiled pBSoriT DNA as the substrate were performed as described under "Experimental Procedures" in the presence of 75 mM NaCl. The products were resolved on a 0.8% agarose gel that was stained with EtBr (0.5 g/ml). Where indicated, TraIp was included at 15 nM, TraYp was included at 200 nM, and IHF was included at 40 nM. The position of the supercoiled DNA substrate (sc) and nicked DNA product (nicked) are indicated.
while the third was added in increasing concentrations. Production of nicked DNA was half-maximal, with TraIp present at a concentration of 7 nM, which is stoichiometric to the substrate (Fig. 4B, closed circles). Production of nicked DNA was half-maximal at a TraYp concentration of ϳ150 nM and an IHF concentration of ϳ20 nM (Fig. 4, A and C, closed circles). The IHF value is in reasonably close agreement with the reported apparent K D for binding to the IHF A site in oriT, whereas the value for TraYp is approximately 4-fold higher than the apparent K D for binding to sbyA (see "Experimental Procedures").
TraIp, TraYp, and IHF Nick a Linear DNA Substrate-TraIp, in the absence of other proteins, fails to catalyze a siteand strand-specific transesterification reaction at an oriT sequence that is on either a relaxed plasmid or linear duplex DNA (10,11). To determine whether this DNA substrate could be specifically nicked when IHF and TraYp were present, various combinations of TraIp, TraYp, and IHF were incubated with a 545-bp linear oriT-containing DNA substrate (Fig. 5). TraIp alone did not nick the linear DNA substrate (Fig. 5, lane  4). The addition of either IHF (Fig. 5, lane 6) or TraYp (Fig. 5,  lane 7) had no effect on this reaction. When all three proteins were incubated with this substrate, a portion of the DNA substrate was nicked at a specific site on a specific strand (Fig. 5,  lane 8). If the site nicked in this reaction is identical to the site nicked in vivo, the nicked DNA product is expected to be 155 nucleotides in length. Using a 5Ј end-labeled linear substrate, the cleavage site was mapped and found to occur at the exact location nicked in vivo (data not shown). Thus TraYp, IHF, and TraIp together were able to catalyze oriT-specific nicking of a linear DNA substrate.
To determine the optimal concentration of each protein required to nick the linear DNA substrate, a series of titration experiments was performed. This analysis was similar to that described above using the supercoiled plasmid substrate. The concentration of each protein required for optimal nicking using the linear nicking assay was similar to that determined in the supercoiled nicking assay, although the extent of the reactions differed (see below). Production of nicked DNA was half-FIG. 4. Titrations to determine optimal concentrations of TraIp, TraYp, and IHF. Nicking reactions using supercoiled pBSoriT DNA were as described for Fig. 3. Panel A, TraIp was held at a fixed concentration of 45 nM, IHF was held at a fixed concentration of 75 nM, and TraYp was varied from 6.8 nM to 1.4 M as indicated. Panel B, TraYp was present at a fixed concentration 680 nM, IHF was present at a fixed concentration of 75 nM, and the TraIp concentration was varied from 1 to 500 nM as indicated. Panel C, TraYp was present at a fixed concentration 680 nM, TraIp was present at a fixed concentration of 45 nM, and IHF was varied from 0.76 to 100 nM as indicated. Reactions were initiated by the addition of TraIp. The data presented represent the average of three or more independent determinations. E, data from reactions using the linear DNA substrate; q, data from reactions using the supercoiled DNA substrate. Error bars represent the standard deviation about the mean. maximal at a TraIp concentration of 4.5 nM, which is a 10-fold molar excess over substrate molecules (Fig. 4B, open circles). Production of nicked DNA was half-maximal at a TraYp concentration of 25 nM and an IHF concentration of 6 nM (Fig. 4, A  and C, open circles). In this case, the values for both IHF and TraYp compare well with reported K D values for the binding sites in oriT. High concentrations of any of the three proteins inhibited nicking of the linear substrate. The results from this series of experiments are in reasonable agreement with those obtained from the titrations performed using the supercoiled DNA substrate.
Kinetics of the Transesterification Reaction-The kinetics of the transesterification reaction were determined in the presence of TraYp and IHF using both the linear and supercoiled DNA substrates. The reactions were initiated by the addition of TraIp to an equilibrated reaction mixture containing substrate DNA, TraYp, and IHF. Presumably, both IHF and TraYp bind their respective binding sites in oriT during the preincubation. The reactions proceeded linearly for approximately the first 5 min and were Ͼ90% complete in 15 min (data not shown). These kinetics are similar to those observed when TraIp alone cleaves a single-stranded DNA substrate. 2 The steady-state amount of nicked DNA observed in each case was below 100%. The reaction using the linear substrate achieved a plateau at 70% nicked, while the reaction using the supercoiled substrate only reached a level of 30% nicked. This likely reflects an equilibrium between nicked and ligated species expected in transesterification reactions (see "Discussion").
Nicking oriT Deletion Mutants-The oriT region is operationally defined as the ϳ300 bp of cis-acting DNA sufficient to direct mobilization of a plasmid by F transfer factors (27). The region includes the nic site, two IHF binding sites (IHF A and IHF B) (18), a TraMp binding site (sbmC) (28), and two TraYp binding sites (sbyA and sbyC) (15, 17) (see Fig. 6). The site sbyC is coincident with IHF A, and presumably both sites cannot be bound by their respective proteins simultaneously. In addition, there are two intrinsic bends present in oriT, each bending the DNA by about 50°C (18). Fu et al. have shown in vivo that elimination of IHF B, the second intrinsic bend, sbmC, and half of sbyA resulted in reduction of the transfer efficiency, with little effect on nicking at oriT (26). To determine which of these sequence features were required for our in vitro reaction, oriT deletion mutants were used as substrates in the supercoiled nicking assay. An advantage of the in vitro assay is the ability to quantify the nicking efficiency of the mutants.
Plasmid pXRD620⌬87 contains bp 1-285 of oriT (using the numbering system of Frost et al. (25)), including nic, IHF A, sbyA, and sbmC, but eliminating IHF B (Fig. 6). Plasmid pXRD620⌬104 contains bp 1-237, eliminating sbmC and one of the intrinsic bend sequences in addition to IHF B. Plasmid pXRD620⌬79 contains bp 1-222, eliminating approximately one-half of sbyA in addition to the deletions described for pXRD620⌬104. When either pXRD620⌬ 87 or pXRD620⌬104 was incubated in the three-protein nicking reaction, the relative amount of nicked DNA formed was comparable with the amount of nicked DNA formed when the substrate was a fully intact oriT sequence. When plasmid pXRD620⌬79 was used, the amount of nicked DNA formed was about 75% of that observed when a plasmid containing the wild-type oriT was used. Elimination of sbmC and the second intrinsic bend sequence had no effect on the in vitro nicking assay reconstituted with three proteins. Also, this experiment shows that the IHF B site is not required for the reaction. The TraYp binding site sbyA can apparently be partially eliminated without loss of nicking competence. However, this plasmid was nicked with a somewhat lower efficiency. Importantly, control experiments indicate that nicking of the pXRD620⌬79 plasmid was IHFand TraYp-dependent (data not shown). In addition TraYp is still able to bind the truncated oriT region of this plasmid, although with reduced affinity (data not shown). DISCUSSION We, and others, have previously reported that TraIp is able to catalyze the site-and strand-specific nicking reaction required to initiate DNA transfer during F plasmid-mediated bacterial conjugation (10,11). The in vitro reaction catalyzed by TraIp is, in fact, a transesterification reaction that requires a supercoiled DNA substrate containing oriT, MgCl 2 , and a molar excess of TraIp (14). A linear DNA substrate or a relaxed, circular DNA substrate cannot be nicked by TraIp. In this communication, we note that this reaction also requires a relatively low ionic strength. In the presence of greater than 75 mM NaCl, the reaction catalyzed by TraIp in the absence of additional proteins is almost undetectable. However, under these conditions, TraIp-catalyzed nicking is greatly stimulated 2 J. A. Sherman, unpublished results.
FIG. 6. Nicking reactions using partial oriT deletion mutants. A partial physical map of the oriT region from F is shown at the top, and base pair coordinates are shown at the bottom. The binding sites for IHF (IHFA and IHFB), TraYp (sbyA), and TraMp (sbmC) are shown. In addition, the nic site is indicated by an arrow and intrinsic sequence-dependent bends have been indicated by carats (16,18,25). Promoters and their directionality for gene X and traM are indicated. Nicking reactions using the indicated supercoiled plasmid (10 nM) were performed as described under "Experimental Procedures" in the presence of 75 mM NaCl. The plasmids have been described in the text. All reactions contained 50 nM TraIp, 204 nM TraYp, and 75 nM IHF and were initiated by the addition of TraIp. Reaction products were resolved on a 0.8% agarose gel that was stained with EtBr (0.5 g/ml). The amount of nicked product formed using each plasmid is indicated relative to the amount of nicked product formed using pBSoriT DNA in a control reaction run in parallel. The data presented represents the average of three independent experiments.
when TraYp and IHF are both present in the in vitro reaction. This result is significant for two reasons. First, it establishes a biochemical role for TraYp in the initiation of conjugative DNA transfer. Previous genetic studies have suggested that TraYp is required for the formation of the site-and strand-specific nick that initiates DNA strand transfer (4). However, a biochemical role for this protein had not been elucidated. The data presented here suggest that TraYp plays an integral role in helping to recruit TraIp to the nic locus. Secondly, the results presented here reveal a critical role for the host-encoded IHF in the process of initiating conjugative DNA strand transfer. Thus a biochemical role for the previously described IHF binding sites in oriT (18) is revealed. It is important to note that both IHF and TraYp are required to stimulate the reaction catalyzed by TraIp; neither protein alone is sufficient. Moreover, extended titrations of both IHF and TraYp suggest that an increased concentration of one protein cannot compensate for the absence of the other protein. 3 This is consistent with the data obtained in the deletion studies (see below).
The direct demonstration that TraYp, in conjunction with IHF, is required for site-and strand-specific nicking catalyzed by TraIp confirms a biochemical role for TraYp in generating the nicked DNA strand that is transferred to the recipient cell. Furthermore, the concentration of TraYp required to observe half-maximal nicking of the linear DNA substrate (approximately 25 nM), in the presence of saturating concentrations of IHF and TraIp, is consistent with binding to the site previously identified as sbyA (15). Half-maximal nicking of the supercoiled substrate is observed at a TraYp concentration of approximately 150 nM. Apparently, alterations of the helical structure in supercoiled DNA reduce the affinity of TraYp for sbyA. These data suggest that TraYp binds independently to this site (as no binding cooperativity is observed between IHF and TraYp) and that sbyA must be occupied by TraYp in order for TraIp to bind and nick at the nic locus. Experiments using deletion mutants that encroach upon oriT from the right further underscore the importance of TraYp binding at sbyA for efficient nicking at oriT (26). In the in vitro experiments presented here, removal of the IHF B binding site resulted in a reaction that was still dependent on both TraYp and IHF. This indicates that IHF is bound at IHF A, presumably occluding sbyC, and therefore TraYp must be bound at sbyA. Also, a deletion that removed approximately one-half of the sbyA binding site reduced the efficiency of the nicking reaction. The affinity of TraYp for this truncated site was also slightly reduced, 3 further supporting the notion that TraYp must be bound to sbyA to help recruit TraIp to the nic locus.
The biochemical role played by TraYp in the nicking reaction was previously unrecognized due to the absence of IHF in reconstituted nicking reaction mixtures. Tsai et al. (18) demonstrated the presence of two binding sites for IHF within the oriT region using direct footprinting studies. The IHF A site lies between the nic locus and the TraYp binding site. This IHF binding site has a higher affinity for IHF than the IHF B binding site, which is 50 bp distal to the TraYp binding site. Protein titration experiments and deletion studies support the idea that binding of IHF to the IHF A binding site, and not IHF B, is critical for recruiting TraIp to the nic locus. The concentration of IHF required for half-maximal nicking (ϳ20 nM) in the presence of saturating concentrations of TraYp and TraIp is consistent with the occupation of the IHF A site by IHF. Moreover, deletion of the IHF B binding site has no effect on the in vitro nicking reaction as demonstrated here and had no effect on nicking observed in an in vivo assay (26). We suggest that IHF and TraYp bind independently to their respective sites in oriT and, under conditions of increased ionic strength, help recruit TraIp to the nic locus.
We envision two mechanisms by which IHF and TraYp might act to stimulate the site-and strand-specific nicking reaction catalyzed by TraIp. In one case, the two proteins might act to distort the DNA helix, perhaps to create a singlestranded DNA binding site for TraIp, at the nic locus. In support of this view, we have shown that TraIp is able to catalyze site-specific nicking of single-stranded DNA (13) while the protein does not specifically bind double-stranded DNA (7). Furthermore, TraIp can specifically nick the oriT region at low ionic strength, a condition that would favor the existence of single-stranded DNA character in a supercoiled DNA substrate. This mechanism is similar to that proposed for the action of DnaA protein at the origin of DNA replication (29). The second mechanism envisions protein-protein interactions that help to assemble a competent relaxosome at oriT. Both IHF and TraYp are known to bend the DNA when bound to their respective binding sites (18,30). In addition, two sequence-directed bends have been localized in oriT (18). Together these factors might alter the overall conformation at oriT to bring the TraYp binding site into juxtaposition with the putative TraIp binding site. This could help to load TraIp at the nic locus and would be consistent with the notion of an interaction between TraYp and TraIp. At present we cannot distinguish between these two possibilities. However, the topology of the starting DNA molecule does not seem to contribute to the specificity of the reaction since both supercoiled and linear DNA molecules can be specifically nicked in the presence of all three proteins. Thus the requirement for a supercoiled substrate is relieved when both TraYp and IHF are present in the reaction mixture. This may have important implications for the termination of DNA strand transfer. It is interesting to note that a higher fraction of the substrate is converted to nicked product when a linear DNA substrate is used as compared with a supercoiled DNA substrate. Since TraIp is able to catalyze both a nicking reaction and a ligation reaction (13), we propose that an equilibrium exists between the nicked and ligated states of the substrate in any TraIp-catalyzed nicking reaction and that the equilibrium depends on reaction conditions. In the in vitro system we have described, a denaturatant and a protease are added to stop the reaction. These additions cause TraIp to release the DNA substrate in whichever state it exists. The difference we see in the fraction of nicked product between the two DNA substrates, therefore, must be due to different equilibria present in the two reactions. The linear substrate could be in the nicked state a higher percentage of the time due to a decreased stability of the relaxosome complex on this DNA substrate. We also note that this is the first reconstituted relaxation reaction for which nicking of a linear DNA substrate has been observed.
In summary, site-and strand-specific nicking at the oriT locus requires binding of both TraYp and IHF to their respective binding sites within oriT. Importantly, the IHF B binding site and the TraMp binding site do not seem to be important for nicking at oriT. This latter conclusion is now well supported by both in vivo and in vitro data. Presumably the binding of both IHF and TraYp help to direct the binding of TraIp to the nic locus. Precisely how this is accomplished remains to be determined. We also note that the results presented here are consistent with those recently reported by Inamoto et al. (31). These authors performed a similar series of experiments using the traI and traY gene products from the related plasmid R100 and reached similar conclusions. manuscript, Dr. Dan Bean and Bob Brosh for stimulating discussions, and Susan Whitfield for preparation of the figures.