(Received for publication, December 23, 1994; and in revised form, May 25, 1995)
From the
The TraI protein encoded by plasmid R100 was purified in a large scale by monitoring the strand- and site-specific nicking activity at the origin of transfer, oriT. The N-terminal amino acid sequence of the purified protein was identical to that deduced from the DNA sequence of an open reading frame encoding TraI. The TraI protein is a DNA helicase which is highly processive and unwinds DNA in the 5` to 3` direction. The Stokes radius and the sedimentation coefficient for the TraI protein in 200 mM NaCl indicate that the protein is a rod-shaped monomer, whose native molecular weight is 186,000. Chemical cross-linking analysis revealed that there exist more dimers of TraI under the low salt conditions, under which both nicking and unwinding reactions catalyzed by TraI are the most efficient, indicating that the TraI protein is functionally active in a dimer form. TraI hardly introduced a nick into the linearized plasmid DNA and only slightly into the relaxed closed circular DNA, indicating that TraI requires superhelical structure of substrate DNA for the nicking reaction. Deletion analysis in the oriT region revealed that a particular region of 54 base pairs containing oriT is required for the nicking reaction.
Conjugation is the process in which DNA is transferred from one bacterial cell harboring a sex factor plasmid, such as F or R100, to another by cell-to-cell contact (for the most recent review, see Frost et al.(1994)). One of the initial events in DNA transfer is the strand- and site-specific nicking at the origin of transfer, oriT, by the plasmid-specified endonuclease that is the traI gene product whose molecular mass is estimated to be about 192 kDa from the nucleotide sequence (Yoshioka et al., 1990; Bradshaw et al., 1990). The TraI protein encoded by plasmid F or R100 has been purified by monitoring its ATPase activity or by its molecular size, respectively. The TraI proteins purified introduce the strand- and site-specific nick, such that it is covalently linked with the 5` end of the nick (Inamoto et al., 1991, 1994; Reygers et al., 1991; Matson and Morton, 1991). The TraI proteins have been known to be DNA helicase (Abdel-Monem and Hoffman-Berling, 1976; Inamoto et al., 1994), which is supposed to unwind the duplex DNA from the nick introduced in the plasmid to provide the single-stranded DNA (Abdel-Monem et al., 1983), which is known to be transferred to the recipient cell (Ohki and Tomizawa, 1968; Rupp and Ihler, 1968).
In this paper, we
purified the TraI protein encoded by R100 by monitoring the site- and
strand-specific endonuclease activity and extensively analyzed the
nicking and unwinding reactions catalyzed by TraI. We show that a dimer
form of the TraI protein is active for both nicking and unwinding
reactions, and that the superhelical plasmid DNA molecules with a
specific region of 54 bp ()are required for the nicking
reaction.
Plasmids, pUC119 (Vieira and Messing, 1987), pSI87-XE1 (Inamoto et al., 1988), and pYY35-1 (Inamoto et al., 1991), were derivatives of pUC18 or pUC19 (Yanisch-Perron et al., 1985) with a DNA segment of plasmid R100. Plasmid pHF11 was constructed by inserting a 236-bp BamHI fragment containing oriT of R100 into pUC18. The BamHI fragment was synthesized by polymerase chain reaction using a pair of primers with a BamHI site which flank the oriT region, followed by digestion of the polymerase chain reaction-amplified fragments with BamHI. The other pHF plasmids used were constructed as described under ``Results.''
The Klenow fragment of DNA polymerase I and T4 polynucleotide kinase were obtained from Takara. Restriction endonucleases, BamHI and HindIII (New England Biolabs), were used. Single-stranded DNA-binding protein (SSB) was obtained from Promega. RNase A and proteinase K were obtained from Sigma and Boehringer Mannheim, respectively. They were used as recommended by their suppliers.
Figure 1:
Specific nicking by the TraI protein. A, schematic representation of the structure of the BamHI fragment (236 bp) with or without a nick. Asterisks indicate the P-labeled 5` ends of DNA strands (thick lines). The fragment was obtained by digestion with BamHI of the pHF11 DNA after treatment of the DNA with the
TraI protein under the conditions described under ``Materials and
Methods.'' Note that the 5` end of the nick is attached by the
TraI protein (open circle) and thus cannot be labeled with
P. B, a 6% polyacrylamide sequencing gel showing
single-stranded DNA fragments generated from the BamHI
fragment after denaturation of the sample DNA with heat. Lane
1, the sample DNA treated with TraI; lane 2, the sample
DNA not treated with TraI. Lanes marked with M indicate sequencing ladders used as size markers. Note in lane
1 that a fragment of 143 nt is generated due to nicking by TraI at oriT (see A).
In every step in these procedures, the relative amount of the TraI protein in each fraction was monitored by electrophoresis in SDS-8% polyacrylamide gels according to Laemmli(1970). The protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin as standard.
Figure 4:
Helicase assay for the TraI protein. A, schematic representation of the substrate DNA used for the
helicase assay. The asterisk indicates the 5` end of the 51-nt
oligonucleotide labeled with P. B, an 8%
polyacrylamide gel, showing the DNA unwinding activity of the TraI
protein. The activity was assayed by monitoring production of the
P-labeled oligonucleotides by the conditions described
under ``Materials and Methods.'' Positions of the substrate and the product are indicated. Lane 1 shows the substrate DNA denatured at 95 °C. Lanes 2-9 show the sample DNA treated with increasing amounts of the TraI
protein (0, 15, 30, 60, 120, 240, 480, and 960 fmol, respectively).
The standard helicase
reaction was performed as follows: 0.5 µl of the substrate (50
fmol) in TE buffer was mixed with 2 µl of 5 buffer N, 0.7
µl of 20 mM ATP, and 5.8 µl of Milli-Q water. After
preincubation for 5 min at 30 °C, the reaction was initiated by
adding 1 µl of the TraI protein in buffer A containing 200 mM NaCl and 20% glycerol. The mixture was incubated at 30 °C for
10 or 5 min, and the reaction was stopped by adding 2 µl of the
stop solution (50 mM EDTA, 0.6% SDS, 40% glycerol, 0.12%
bromphenol blue). The reaction mixture was electrophoresed on a
nondenaturing 8% polyacrylamide gel in TAE buffer (Maniatis et
al., 1982), and the radioactivity of bands was determined using
Bio-Image Analyzer.
Figure 6:
Direction of DNA unwinding by TraI. A, substrate DNA, and
, used to determine the
direction of DNA unwinding by TraI. Asterisks indicate the 5`
ends labeled with
P. B, a 10% polyacrylamide gel,
showing the unwinding activity of the TraI protein. Substrate
or
was incubated with TraI, and the products were analyzed using the
conditions described under ``Materials and Methods.'' Note
that substrate
was unwound, whereas substrate
was not
unwound, demonstrating that TraI travels in the 5` to 3` direction on
single-stranded portion of the substrate and unwinds the
double-stranded DNA.
Using this method of detection of the site- and strand-specific
endonuclease activity of the TraI protein, we tried to purify the TraI
protein in a large scale by column chromatography from the crude lysate
of cells harboring plasmid pYY35-1 (traI) overproducing the TraI protein. The
crude cell lysate contained the TraI protein in an amount greater than
20% of total protein as estimated by SDS-polyacrylamide gel
electrophoresis, but did not show any nicking activity (Table 2).
However, a fraction obtained from phosphocellulose column showed the
nicking activity (Table 2), suggesting that the initial crude
lysate contained either an inhibitor(s) or a nuclease(s) causing
degradation of the substrate DNA. The TraI protein in the
phosphocellulose column fraction could be purified to homogeneity
through MonoQ FPLC, Superdex 200pg, and HiTrap Heparin columns (see Table 2). The specific activity of the TraI protein increased 6.2
times through the purification (Table 2).
In these purification steps, the specific activity of the fraction obtained using MonoQ column was 4.6 times greater than that of the fraction obtained from phosphocellulose column. However, the relative amount of TraI estimated by scanning SDS-polyacrylamide gels increased only 1.1 times (Table 2). This suggests that the phosphocellulose fraction still contained an inhibitor(s) or a nuclease(s). In phosphocellulose, Superdex 200pg or MonoQ column chromatography, the nicking activity corresponded to the single peak of protein mass (see Fig. 2A for the result of Superdex 200pg column chromatography). In HiTrap Heparin column chromatography, however, the profile of the nicking activity showed two peaks, which corresponded to those in the profile of protein concentration (Fig. 2B). We assume that this is due to multimerization of the TraI protein, as will be discussed later.
Figure 2: Column chromatography of the TraI protein. A, Superdex 200pg column. B, HiTrap Heparin column. The nicking activity (closed circles) and protein concentration (open circles) of each fraction were determined as described under ``Materials and Methods.''
We carried out amino acid sequencing analysis of the purified
TraI protein and found that the N-terminal amino acid sequence was
MLSFSVV, which is identical to that deduced from the
nucleotide sequence of an open reading frame for the traI gene
(Yoshioka et al., 1990). This in turn indicates that the N
terminus of the TraI protein is neither blocked nor processed.
We then carried out the sedimentation analysis of the purified TraI protein in the buffer containing 50 mM NaCl, and found that the sedimentation coefficient sifted to 8.9 S (Table 3). This suggests that TraI forms dimers under the low salt conditions. To verify this assumption, we carried out chemical cross-linking analysis and found that TraI monitored by SDS-polyacrylamide gel electrophoresis existed as dimers under the low salt conditions but as monomers under the high salt conditions (Fig. 3).
Figure 3: Cross-linking analysis of the TraI protein. A, an SDS-5% polyacrylamide gel stained with Coomassie Brilliant Blue, showing the TraI protein chemically cross-linked under the various concentrations of NaCl. Lane 1, TraI not cross-linked; lanes 2-8, cross-linked TraI at the salt concentration indicated at the bottom. Cross-linking was carried out as described under ``Materials and Methods.'' Positions of monomers (186 kDa), dimers (372 kDa), and multimers of TraI are indicated on the left side of the gel. Molecular size standards used were ferritin (440 kDa), catalase (230 kDa), aldolase (158 kDa), and bovine serum albumin (66.2 kDa). B, relative amounts of monomers, dimers, and multimers of TraI under the various concentrations of NaCl, as determined by densitometric scan of the gel shown in A using EPSON GT-6000 scanner and NIH image.
Figure 5: Unwinding reaction (A) and nicking reaction (B) by TraI. a, time course of the unwinding or nicking reaction. Concentrations of TraI and substrate used were 11 and 5 nM, respectively. b, stoichiometry of the unwinding or nicking reaction. c, effect of the NaCl concentration on the unwinding or nicking reaction. Concentrations of TraI and substrate used were 22 and 5 nM, respectively. The helicase and nicking activities were assayed by the conditions described under ``Materials and Methods.''
Lineweaver-Burk plotting of the data gave V of 0.47 nM/min and K
of 6.6 nM at 30 °C,
indicating efficient binding of TraI to the substrate DNA. The value of V
corresponds to an unwinding velocity of 290
bp/s, which means that the unwinding reaction is immediately completed
once the reaction starts. From these values, we calculated k
(turnover) to be 0.12/min and k
/K
to be 1.9
10
M
min
. The values indicate that TraI does not
rapidly turn over. These results show that the TraI helicase is highly
processive.
We examined several parameters for the unwinding
reaction. The unwinding reaction occurred most efficiently at 37
°C. Optimal pH was 7.2. The unwinding reaction absolutely required
Mg and ATP. The optimal concentration of
Mg
is 5 mM, and that of ATP is 4 mM
as far as we measured the activity at the concentrations from 0.25 to 4
mM. We also examined the effect of the single-stranded
DNA-binding protein, SSB, on the unwinding reaction. SSB promoted the
unwinding reaction slightly, about 5%, at the concentration of 16
nM tetramer (3.2 times over substrate), but inhibited the
reaction to a certain extent at the concentrations of over 120 nM (24 times over substrate); SSB inhibited the reaction by 31% at
400 nM (80 times over substrate) and by 47% at 1.6 µM (320 times over substrate).
We also determined the direction of
DNA unwinding by TraI using the substrate, or
, which is a
24-bp duplex with the 5`- or 3`-overhang of 18 nt, respectively (Fig. 6A). TraI unwound substrate
with the
5`-overhang very efficiently, but unwound substrate
with the
3`-overhang very poorly (Fig. 6B). This indicates that
TraI unwinds DNA in the 5` to 3` direction. This agrees with the
direction of unwinding by TraI (Helicase I) encoded by F (Lahue and
Matson, 1988).
We then examined several parameters for
the nicking reaction. The nicking reaction occurred most efficiently at
37 °C. Optimal pH was 7.6. The optimal concentration of
Mg was 10 mM. Unlike the unwinding reaction,
the nicking reaction occurred in the absence of ATP. ATP rather
inhibited the nicking activity, probably because ATP enhanced the
helicase activity that leads to movement of TraI on DNA, resulting in
inhibition of the nicking reaction. We also examined the effect of SSB
on the nicking reaction. Interestingly, SSB inhibited the nicking
reaction to 33% at 13 nM (5.4 times over substrate) and to 7%
at 210 nM (87 times over substrate).
Figure 7: DNA substrate required for the nicking reaction. A, nicking at oriT on superhelical DNA (closed circles), relaxed closed circular DNA (open circles), or linearized DNA (closed squares) of plasmid pHF11. The nicking activity was assayed as described under ``Materials and Methods.'' B, nicking of superhelical pHF11 DNA in the presence of ethidium bromide. The nicking reaction was initiated by adding 2 µl of the TraI protein (477 fmol/µl) after addition of ethidium bromide to the reaction mixture and preincubation for 12 min.
As described above, the sequence recognized by TraI could not be determined using the linear fragments of plasmid DNA. To define the region required for nicking by TraI, therefore, we constructed a set of plasmids with a deletion in the oriT region (see Fig. 8) and examined them to see whether TraI introduces a nick or not. As shown in Fig. 8, a particular region of 54 bp containing oriT was found to be essential for nicking. This indicates that this 54-bp region is recognized by TraI for the nicking at oriT.
Figure 8: The essential region for nicking by TraI. A critical portion of the oriT region is shown at the top. Numbers are nucleotide positions defining the 3` end of the nick as +1, and the 5` end of the nick as -1. Inverted repeat sequences are indicated by pairs of arrows between two DNA strands. The IHF-binding site, ihfA (Inamoto et al., 1990), and the TraY-binding site, sbyA (Inamoto and Ohtsubo, 1990), are shown by hatched and open boxes, respectively. The pHF plasmids carry a DNA segment from the oriT region of R100 (thick lines). These DNA segments were synthesized by polymerase chain reaction using oligonucleotide primers with a BamHI site. The polymerase chain reaction-amplified fragments were digested with BamHI and cloned into the BamHI site of pUC18. The results of nicking by TraI are shown on the right side of the panel.
In this paper, we have shown that the TraI protein of R100 is a rod-shaped molecule with molecular weight 186,000. This is consistent with the result obtained by electron microscopy that the protomer of TraI protein of plasmid F is rod-shaped (Abdel-Monem et al., 1977). We have also shown here that the TraI protein forms dimers under the low salt conditions and monomers under the high salt conditions, and that dimers of TraI are active in both nicking and unwinding reactions. Previous stoichiometric analysis has suggested that a multimer consisting of about 70 to 90 monomers of TraI of F shows the highest helicase activity (Kuhn et al., 1978; Benz and Muller, 1990), and that 10 molecules of TraI of F per DNA molecule show the highest helicase activity although substantial activity was detected in the reaction involving a 1:1 ratio of enzyme molecules to DNA molecules (Lahue and Matson, 1988). These results differ from ours obtained for TraI of R100. It is noted here, however, that several other helicases, except one, form either dimers or hexamers (Lohman, 1992). Rep helicase and helicase II (UvrD) have been shown to be dimerized upon binding to either single-stranded or duplex DNA (Wong et al., 1992; Runyon et al., 1993). This reminds us the result that in the purification procedure of TraI protein, chromatography using the HiTrap Heparin column showed two peaks, although chromatography using other columns showed a single peak (see Fig. 2). This may suggest that TraI was dimerized upon binding to heparin, a DNA analog.
We have shown here that the TraI protein introduces the nick into only a little over half of the substrate DNA even upon long incubation and/or addition of an excess amount of the TraI protein. It is possible that the nicking reaction requires some factors, which are not present in the reaction mixture. Such factors may include the traY gene product and the integration host factor IHF, which have been recently shown to stimulate the nicking reaction catalyzed by TraI (Inamoto et al., 1994) by binding to the sites (sbyA and ihfA) located immediately adjacent to the nicking site oriT (Inamoto and Ohtsubo, 1990; Inamoto et al., 1990). It might also be possible that TraI possesses the topoisomerase activity to convert superhelical DNA to relaxed closed circular DNA, which was shown in this paper to be poorly nicked by the TraI protein. However, the TraI protein has no topoisomerase activity, since the relaxed circular DNA molecules were not detected in an agarose gel containing chloroquine after electrophoresis of the sample treated with TraI (data not shown). We cannot, however, exclude the possibility that the nicked molecules with TraI covalently attached are still in a superhelical DNA form and can be readily converted to the superhelical DNA molecules without TraI, leading to an equilibrium state between two kinds of superhelical DNA molecules with and without TraI.
We have
recently found that the TraI protein has the single-stranded DNA
binding activity and cleaves the single-stranded DNA at oriT. ()This DNA cleaving activity is inhibited by SSB. These
findings lead us to assume that the oriT region in the
double-stranded DNA molecules melts locally, providing a
single-stranded DNA portion which is cleaved by TraI. This assumption
may be supported by our present result that the nicking reaction is
strongly inhibited by SSB.
The nicking reaction occurs most
efficiently under the low salt conditions, but it hardly occurs at high
salt (100 mM NaCl or more), as described in this paper. The
cleavage reaction of the single-stranded DNA by TraI occurs most
efficiently under the low salt conditions like the nicking reaction,
but it still occurs even at the NaCl concentration 100 mM unlike the nicking reaction. It is possible that the
superhelical DNA, that is the substrate for the nicking reaction,
cannot be melted locally under the high salt conditions, thus not
providing any single-stranded DNA portion which is bound and cleaved by
the TraI protein. Alternatively, dimers of TraI formed under the low
salt conditions may form a DNA-protein complex at the partially melted
region around oriT which leads to the cleavage reaction, but
monomers formed under the high salt conditions may not form such a
complex.