(Received for publication, June 5, 1995; and in revised form, September 9, 1995)
From the
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 K 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 5`-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 (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 extraspeciesspecific 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 strand-specific 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, 7, 8, 9) . More recently, we and others have shown that TraIp also contains the catalytic site responsible for site- and strand-specific 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 DNA-binding protein with three known binding
sites(15, 16, 17) . Two of these binding
sites are located within oriT within 100 bp ()of nic, the site that is nicked in vivo to initiate
strand transfer. The position of these sites suggests the possibility
of protein-protein 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 oriT-containing 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.
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 5.5 cm) heparin-agarose (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
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
-70 °C. Purified TraIp was greater than 90% homogeneous as
judged by electrophoresis in the presence of SDS (Fig. 1A).
Figure 1:
SDS-polyacrylamide gel
analysis of purified TraIp, TraYp and IHF. Panel A,
approximately 1 µg of purified TraIp (helicase I) was resolved on
an 8% polyacrylamide gel run in the presence of SDS. Markers (lane
M) were high range SDS-PAGE standards (Bio-Rad): myosin (200 kDa),
-galactosidase (116 kDa), phosphorylase B (97.4 kDa), and bovine
serum albumin (66 kDa). Panel B, 1.9 µg of purified IHF
and 2 µg of TraYp were resolved on a 12% polyacrylamide gel run in
the presence of SDS. Markers (lane M) were low range SDS-PAGE
standards (Bio-Rad): phosphorylase B (97.4 kDa), bovine serum albumin
(66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean
trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The gels were
run according to the procedure of Laemmli (32) and stained with
Coomasie Brilliant Blue.
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 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
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
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
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
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 >90% 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 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 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
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
of TraYp for sbyA was 36.6 nM.
Plasmids containing oriT deletions were
kindly provided by Dr. Richard Deonier (University of Southern
California). Plasmid pXRD620 87 contains bp 1-285 of oriT (using the numbering system of Frost et
al.(25) ) inserted into the BamHI site of pUC8;
pXRD620
104 contains bp 1-237; pXRD620
79 contains bp
1-222(26) .
Nucleotides were purchased from
Pharmacia-P/L Biochemicals. [-
P]dCTP was
obtained from Amersham, Corp. DNA concentrations are expressed in terms
of mol of DNA molecules.
Figure 2:
The effect of increasing NaCl
concentration on nicking reactions catalyzed by TraIp alone or in the
presence of TraYp and IHF. Nicking reactions using supercoiled pBSoriT
DNA as the substrate were performed as described under
``Experimental Procedures'' and included the indicated
concentrations of NaCl. Reaction mixtures contained either 14.2
nM purified TraIp alone () or 14.2 nM purified
TraIp, 147.5 nM purified TraYp, and 70 nM purified
IHF (
). The fraction of input plasmid that was nicked was
quantified as described under ``Experimental Procedures.''
There was a small (<5%) 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.
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.
Figure 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 µg/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.
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, 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 150 nM and an IHF concentration
of
20 nM (Fig. 4, A and C, closed circles). The IHF value is in reasonably close
agreement with the reported apparent K
for binding
to the IHF A site in oriT, whereas the value for TraYp is
approximately 4-fold higher than the apparent K
for binding to sbyA (see ``Experimental
Procedures'').
Figure 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 µM 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. , data from reactions using the linear
DNA substrate;
, data from reactions using the supercoiled DNA
substrate. Error bars represent the standard deviation about
the mean.
Figure 5:
TraYp and IHF promote TraIp-catalyzed
nicking of a linear DNA substrate. Nicking reactions using a linear oriT-containing DNA substrate were performed as described
under ``Experimental Procedures'' in the presence of 75
mM NaCl. The P-labeled DNA substrate was a 545-bp XbaI/SalI restriction fragment prepared and labeled
as described under ``Experimental Procedures.'' The products
were resolved in a 8% polyacrylamide, 8 M urea denaturing gel
and visualized by autoradiography. Where indicated, TraIp was included
at a concentration of 35.5 nM, TraYp was included at a
concentration of 270 nM, and IHF was included at a
concentration of 500 nM. The position of the linear DNA
substrate and the nicked product are indicated on the left.
The markers (lane M) were BioMarker(TM) LOW (BioVentures,
Inc.) labeled with [
-
P]dCTP as suggested by
the manufacturer.
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-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 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.
Figure 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 µg/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.
Plasmid pXRD62087 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 IHF- and 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).
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,
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 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. (
)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, 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 (20
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 single-stranded 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.