Swapping single-stranded DNA sequence specificities
of relaxases from conjugative plasmids F and R100
Matthew J. Harley* and Joel F. Schildbach†
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Communicated by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, August 6, 2003 (received for review May 25, 2003)
Conjugative plasmid transfer is an important mechanism for diversi-
fying prokaryotic genomes and disseminating antibiotic resistance.
Relaxases are conjugative plasmid-encoded proteins essential for
plasmid transfer. Relaxases bind and cleave one plasmid strand site-
and sequence-specifically before transfer of the cleaved strand.
TraI36, a domain of F plasmid TraI that contains relaxase activity,
KDand high sequence specificity. Despite 91% amino acid sequence
identity, TraI36 domains from plasmids F and R100 discriminate
between binding sites. The binding sites differ by 2 of 11 bases, but
both proteins bind their cognate site with three orders of magnitude
higher affinity than the other site. To identify specificity determi-
nants, we generated variants having R100 amino acids in the F TraI36
background. Although most retain F specificity, the Q193R?R201Q
The reverse switch (R193Q?Q201R) in R100 TraI36 confers a wild-type
base contributions to recognition suggests that the specificity differ-
ence derives from multiple interactions. The F TraI36 crystal structure
shows positions 193 and 201 form opposite sides of a pocket within
the binding cleft, suggesting binding involves knob-into-hole inter-
actions. Specificity is presumably modulated by altering the compo-
sition of the pocket. Our results demonstrate that F-like relaxases can
switch between highly sequence-specific recognition of different
sequences with minimal amino acid substitution.
gative plasmid directs transfer of a copy of itself, in single-stranded
form, to a recipient cell (reviewed in refs. 1–3). Despite the
sequence diversity of conjugative plasmids that have been studied,
many elements of the transfer process are conserved (3), including
the involvement of relaxases. Relaxases, also called mobilization
proteins or nickases, are essential for conjugation (4–6). These
proteins bind and cleave one DNA strand at the plasmid origin of
transfer (oriT). The cleaved strand is transferred to the recipient.
Relaxases act as part of the relaxosome, a complex of multiple
proteins and plasmid DNA (6–8). The relaxosome may serve to
generate the single-stranded DNA (ssDNA) substrate that the
relaxase targets. Relaxases cleave ssDNA in a Mg2?-dependent
transesterification reaction that proceeds through a stable phos-
photyrosyl intermediate (9–15). At the end of conjugative transfer,
Relaxases exhibit sequence specificity for their cognate oriT
sequences (9, 11, 12, 20–26). We previously demonstrated, by using
the 36-kDa relaxase domain of F TraI (TraI36), an N-terminal
330-aa fragment of F factor TraI (27), that this specificity can be
remarkable. F TraI36 binds a single-stranded F oriT sequence with
a subnanomolar KD, and some single-base substitutions over a
10-base region can reduce affinity by between 10- and 10,000-fold
(24). In vivo, this specificity is reflected in the poor efficiency with
which F TraI mobilizes plasmids containing the TraI-binding site
for the highly homologous R100 plasmid, despite a difference of
only two bases (24, 28). To better understand the basis of ssDNA
that contribute to ssDNA recognition. Here we demonstrate that
despite the 91% amino acid sequence identity shared by F factor
between prokaryotes. During bacterial conjugation, a conju-
and R100 TraI36, the two proteins have easily detected differences
in specificity. We then describe the results from a panel of TraI36
variants that identify specificity determinants. We found that just
two amino acid residue differences account for the two base
difference in ssDNA binding and cleavage specificity.
Materials and Methods
Protein Engineering and Purification. Primersequencesareavailable
on request. All cloned gene segments and mutations were con-
firmed by DNA sequencing. The F TraI36 expression construct
(pET24a-traI36) was engineered previously (27). For R100 TraI36
expression, the N-terminal 331-codon region of R100 traI was PCR
amplified. Primers encoded an NdeI site overlapping the start
codon and an EcoRI site after a stop codon engineered at codon
332. The NdeI?EcoRI-digested PCR product was ligated into
NdeI?EcoRI-digested pNEB193 (New England Biolabs), trans-
traI36 gene was excised from pNEB193, ligated into NdeI?EcoRI-
digested pET24a(?) (Novagen), and transformed into strain XL10
Gold. The 3?-most codon was removed by PCR as described (27).
Gene fragments for chimera R100?F?F were generated by
NdeI?PstI digestion of the TraI36 expression vectors, purified by
electrophoresis through low melt agarose, isolated by using the
Wizard PCR Preps DNA Purification System kit (Promega), and
ligated. The R100 insert fragment for chimera F?F?R100 was
PCR-amplified by using R100 traI36 as template, and cloned into
purified StuI?EcoRI-digested pET24a-traI36. Constructs were
transformed into strain TB1. An unintended M278T mutation in
chimera F?F?R100 was corrected by using the QuikChange site-
directed mutagenesis kit (Stratagene).
Single, double, and triple mutations were generated by using the
QuikChange kit. An ‘‘F’’ or ‘‘R’’ preceding the variant description
indicates F or R100 background, respectively. The F-Q193R?
R201Q variant was PCR-generated by using the F-Q193R traI36
variant as the template. Variants F-E153D?Q193R?R201Q and
F-I185S?Q193R?R201Q were PCR-generated from F-Q193R?
R201Q, R-R193Q?Q201R from R-Q201R, and R-D153E?
R193Q?Q201R from R-R193Q?Q201R.
Expression plasmids were transformed into strain BL21(DE3).
Proteins were expressed and purified as described for wild type F
TraI36 (27), and concentrated by using CentriCon10 or Centricon
Plus-20 filters (Amicon).
Oligonucleotide Synthesis and Purification. Sequences of oligonucle-
otides used in binding and cleavage assays are shown in Fig. 1.
Oligonucleotides were purchased from Integrated DNA Technol-
incorporated by using TAMRA-CPG columns. Labeled oligonu-
Abbreviations: TraI36, 36-kDa relaxase domain of TraI; ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA; oriT, plasmid origin of transfer; TAMRA, carboxytetra-
*Present address: Department of Plant Pathology, University of California, One Shields
Avenue, Davis, CA 95616.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
September 30, 2003 ?
vol. 100 ?
no. 20 ?
R193Q?Q201R display cleavage characteristics intermediate be-
tween F TraI36 and their R100 TraI36 parent. Both variants
generate more F oriT cleavage product at 100 nM protein than
Q201R (Fig. 6 Upper). Both variants have decreased apparent
activity against the R100 oriT oligonucleotide, relative to R100
TraI36, but still had more apparent activity than F TraI36 (Fig. 6
Lower). R-R193Q and R-Q201R display greater apparent cleavage
despite affinities 200- and 600-fold lower than R-R193Q?Q201R
(data not shown). R-R193Q and R-Q201R have not fully switched
specificities as, unlike wild-type F TraI36, they still cleave the R100
oriT oligonucleotide well.
differences between the binding and cleavage specificities of F and
R100 TraI36 reflect differences in both binding and catalysis. The
ability of a TraI36 protein to cleave F and R100 oriT oligonucle-
R100 oriT site than does F TraI36, and only 4-fold higher affinity
than the R-R193Q?Q201R variant, yet cleaves the R100 oligonu-
cleotide almost to completion at 100 nM, whereas the other two
proteins generate little product at this concentration. Substitutions
cleavage product is accumulated. This is due to dissociation be-
coming more favored relative to the reverse (ligation) reaction
(24). Although an enhanced dissociation rate constant could ex-
the F and R100 variants, it is also possible that oligonucleotides
more readily cleaved. This could be a direct influence of these
amino acids on the catalytic mechanism, or could be indirect,
resulting from their effects on the orientation of other amino acids
or bound DNA.
Implications. Our results indicate that the specificity of F and F-like
relaxases, highly sequence-specific ssDNA-binding proteins, can be
altered with as few as two amino acid substitutions. Similar con-
versions between naturally occurring binding specificities on sub-
stitution of one or a few amino acids have also been reported for
several sequence-specific double-stranded (ds) DNA-binding pro-
teins (34–39). Despite this apparent similarity, our relaxase results
are distinct from those of dsDNA binding proteins for a number of
reasons. First, the stoichiometry of TraI36 binding is 1:1. In
contrast, many of the dsDNA-binding proteins studied are oligo-
meric or are monomers that bind cooperatively to two or more
distinct sites. For these proteins, the use of multiple binding units
amplifies the effect of any single amino acid substitution. Second,
the level of specificity exhibited by F TraI36 suggests that the
binding site has a high degree of surface complementarity to the
DNA. This complementarity is likely to be highly refined and
potentially difficult to alter successfully, especially with few amino
acid changes. Third, although relaxase activity is essential to con-
jugative plasmid transfer, it is not essential for plasmid mainte-
nance. It therefore seems possible that a plasmid could endure a
multistep process that alters relaxase recognition and the oriT
sequence while remaining viable, even if the intermediate muta-
tional steps left the plasmid unable to transfer. In contrast, it seems
less likely that such a process could be tolerated for a DNA-binding
protein that regulates an essential cellular process that when
disrupted could result in cell death. Fourth, TraI has both ssDNA-
binding and -cleavage activities. The particular geometry required
by the enzymatic reaction and the constraints that it would put on
the architecture of the binding and active site might limit the range
of specificities and the manner in which specificity is determined.
The crystal structure of F TraI36 (40) provides some insight into
ssDNA recognition by TraI36. The binding surface of F TraI36
includes a number of pockets or pits ?4 Å deep and 7–10 Å in
diameter, which modeling experiments suggest are capable of
accommodating DNA bases (40). Gln-193 and Arg-201 are located
on opposite sides of one of these pockets (Fig. 7). Arg-201 also
forms part of a second pocket. DNA recognition by TraI36
probably occurs in part through a knob-into-hole fashion, consis-
tent with good surface complementarity and the observed high
sequence specificity. This simple model of interaction, however,
may be insufficient to account for all of the binding data. We
continue attempts at cocrystallization of TraI36 with ssDNA in an
effort to gain further insight into the structural basis of their
We show here that relaxase specificity can be enormously influ-
enced by the amino acids at certain key positions, and substitutions
at these positions can alter specificities dramatically. Similar obser-
vations have been made for a number of dsDNA-binding proteins.
Relaxases differ remarkably from dsDNA-binding proteins, how-
ever, in that the key specificity-determining residues of relaxases
may be frequently located at pockets within their DNA-binding
We thank Dmitri Toptygin for helpful discussions of fluorescence;
Dmitri Toptygin, Daniel Isom, and Christopher Larkin for guidance in
data collection; and Ludwig Brand, Robert Schleif, and Douglas Fam-
brough for use of equipment. This article is based on work funded by
National Science Foundation Grant MCB-9733655 and National Insti-
tutes of Health Grant GM61017.
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www.pnas.org?cgi?doi?10.1073?pnas.2035001100 Harley and Schildbach