(Received for publication, September 8, 1995; and in revised form, January 3, 1996)
From the
The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent. Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides.
Escherichia coli contains two type I DNA
topoisomerases, DNA topoisomerase I (1) and
III(2, 3) . DNA Topoisomerase I (Topo I), ()a 865-amino acid polypeptide(4) , has been
proposed to be involved with the maintenance of the superhelical
density of the bacterial chromosome by acting as an antagonist to DNA
gyrase (reviewed by Wang(5) ). It has been shown to possess an
ATP-independent relaxation activity (1) and is capable of
catalyzing the catenation of nicked, duplex DNA circles (6, 7, 8) and the decatenation of these
singly linked catenanes(6, 9) .
DNA topoisomerase
III (Topo III), a 653-amino acid polypeptide(10) , was
originally purified as a superhelical DNA relaxation activity from
cells containing a deletion of the gene encoding Topo I (topA)(2, 3) . It was subsequently purified
as a potent decatenating activity based on its ability to resolve
plasmid DNA replication intermediates in vitro(11) .
Unlike Topo I, Topo III-catalyzed relaxation of negatively supercoiled
DNA was virtually undetectable under standard assay conditions (10
mM Mg, 50 mM Na
or
K
, 37 °C), requiring high temperature (52 °C)
and low magnesium (1 mM) and monovalent salt concentrations
(<20 mM) to exhibit maximal
activity(2, 3, 11) . The decatenation of
multiply interlinked plasmid DNA dimers and resolution of DNA
replication intermediates catalyzed by Topo III, however, does not
require these extreme conditions and proceeds quite efficiently under
the standard reaction conditions stated above(11) .
Interestingly, Topo I does not catalyze the resolution of DNA
replication intermediates in vitro(12) . This has led
to the suggestion that Topo III may play a role in the decatenation of
newly replicated DNA, whereas Topo I is involved in the maintenance of
the superhelical density of the chromosome(11) .
Analysis of the gene encoding Topo III (topB) indicated that this topoisomerase exhibited striking protein sequence homology to Topo I(10) . Topo I and Topo III, therefore, are an example of two proteins, sharing extensive protein sequence homology, that catalyze distinct reactions. The homology between Topo I and Topo III extends only through the first 600 amino acids of the two polypeptides. The carboxyl-terminal amino acid residues of Topo I and Topo III show no homology, but each has been shown to be involved in substrate binding (13, 14) . The carboxyl terminus of Topo I contains three zinc finger motifs and a high density of arginine and lysine residues(15) . The carboxyl terminus of Topo III does not contain any known motif but, similar to Topo I, contains a high density of clustered, positively charged amino acid residues(14) . Since the carboxyl-terminal residues differ in the two polypeptides, the role that this region plays in topoisomerase-catalyzed relaxation and decatenation was examined. The nature of the differences in the two polypeptides was addressed by comparing the properties of Topo III with a truncation of the enzyme (lacking the putative carboxyl-terminal 49-amino acid residue substrate binding domain(14) ), Topo I, and a hybrid molecule that contained the amino-terminal 605 amino acids of Topo III fused to the carboxyl-terminal 312-amino acid residue substrate binding domain of Topo I.
The two fragments were used in a ligation containing NdeI-BamHI cut pET-3c in order to generate the hybrid gene (pT31Z). A plasmid DNA containing the correct insert was isolated and was then transformed in E. coli BL21.
Figure 1:
Schematic diagram of
Topo 31Z, a Topo III-Topo I chimera. Panel A, the synthesis of
the gene encoding Topo 31Z is described under ``Materials and
Methods.'' Topo 31Z consists of the first 605 amino acids of Topo
III and the carboxyl-terminal 311 amino acids of Topo I (amino acids
553-865). The carboxyl-terminal region of Topo I contains three
zinc finger motifs (15) , whereas the carboxyl-terminal amino
acids of Topo III contain clustered lysine and arginine
residues(14) . Panel B, the amino acid sequences of
Topo I, Topo III, and Topo 31Z are presented. The synthesis of Topo 31Z
resulted in a single change in the carboxyl-terminal residues of Topo
I: Val was changed to a leucine residue (filled
circle) as a result of the cloning
procedure.
Figure 2: SDS-polyacrylamide gel electrophoresis of purified Topo 31Z. Topo 31Z was purified to apparent homogeneity as described under ``Materials and Methods.'' 1.0 µg of the purified enzyme was electrophoresed through a 10% polyacrylamide in the presence of 0.1% SDS(27) . The protein was visualized by staining the gel with Coomassie Blue. Lane 1, Topo 31Z; lane 2, molecular mass markers.
Figure 3:
DNA relaxation assay of Topo III, Topo
III, Topo I, and Topo 31Z. DNA relaxation assays were
performed as described under ``Materials and Methods.'' Upper panel, reactions (25 µl) contained no topoisomerase (lanes 1 and 10), 200 fmol (lane 2), 500
fmol (lane 3), 1000 fmol (lane 4), or 1500 fmol (lane 5) of Topo III
or 50 fmol (lane
6), 100 fmol (lane 7), 200 fmol (lane 8), or 500
fmol (lane 9) of Topo 31Z. Lower panel, reactions (25
µl) contained no topoisomerase (lanes 1 and 14)
or 50 fmol (lanes 2, 6, and 10), 100 fmol (lanes 3, 7, and 11), 200 fmol (lanes
4, 8, and 12), and 500 fmol (lanes 5, 9, and 13) of either Topo 31Z (lanes
2-5), Topo I (lanes 6-9), or Topo III (lanes 10-13). OC, open circle (nicked or
gapped circular DNA). SC, supercoiled (negatively supercoiled
circular DNA).
Figure 4:
Oligonucleotide gel mobility shift assay
of Topo III, Topo III, Topo I, and Topo 31Z. A 45-base
oligonucleotide that contained a strong Topo III binding/cleavage site
(oligonucleotide 45C) was incubated with no topoisomerase (lane
1), or 1.2 pmol (lanes 2, 5, 8, and 11), 3.6 pmol (lanes 3, 6, 9, and 12), or 10.8 pmol (lanes 4, 7, 10,
and 13) of Topo III (lanes 2-4), Topo
III
(lanes 5-7), Topo 31Z (lanes
8-10), or Topo I (lanes 11-13). The reactions
were processed and resolved through a 10% polyacrylamide gel. The
position of the stable topoisomerase-oligonucleotide complex is
indicated. The amount of the topoisomerase-oligonucleotide complex was
determined as described under ``Materials and
Methods.''
The cleavage site specificity of Topo
31Z was also compared with Topo III and Topo I (Fig. 5). A
22-base oligonucleotide containing a subsequence of the 45-base
oligonucleotide was used as a substrate in a topoisomerase-catalyzed
DNA cleavage assay. This oligonucleotide contains distinct Topo III and
Topo I cleavage sites. Topo 31Z-induced cleavage of this substrate
occurs at the strong Topo III site (compare lanes 1 and 2 to lane 4) rather than the Topo I cleavage sites (lane 3). This is consistent with the observation that the
requirements for the relaxation of supercoiled DNA by the hybrid
molecule closely resemble those of Topo III and is also consistent with
the previous observation that Topo III exhibits the same
cleavage site specificity as Topo III, in spite of the removal of its
generalized DNA binding domain(14) .
Figure 5: Determination of the cleavage site specificity of Topo 31Z. Cleavage reactions were performed as described under ``Materials and Methods.'' Reactions contained 1 pmol (lane 1) or 0.1 pmol (lane 2) of Topo 31Z, 0.1 pmol of Topo I, or 0.1 pmol of Topo III. Reaction products were separated by electrophoresis through a 25% polyacrylamide gel in the presence of 50% (w/v) urea as described under ``Materials and Methods.'' NT, nucleotides.
Figure 6:
Analysis of products generated by Topo I,
Topo III, Topo 31Z, and Topo III during oriC replication in vitro. oriC DNA replication
reactions were performed as described previously(12) . Panel A, reactions (12.5 µl) contained no Topo I or Topo
III (lanes 1 and 6) or 12 fmol (lanes 2 and 7), 35 fmol (lanes 3 and 8), 105 fmol (lanes 4 and 9), or 315 fmol (lanes 5 and 10) of Topo I (lanes 2-5) or Topo III (lanes 7-10). Panel B, reactions contained no
Topo 31Z or Topo III
(lanes 1 and 6) or
105 fmol (lanes 2 and 7), 315 fmol (lanes 3 and 8), 945 fmol (lanes 4 and 9), or
2835 fmol (lanes 5 and 10) of Topo 31Z (lanes
2-5) or Topo III
(lanes 7-10).
Fully decatenated form II molecules are indicated as is a late
replication intermediate (LRI) present in the reaction. Panel C, the autoradiographs presented in panels A and B were quantified using a Fuji BAS 1000 phosphor
imager. The percentage of fully decatenated Form II molecules present
in each reaction is plotted as a function of topoisomerase
concentration. Closed circles, Topo III; open
circles, Topo I; closed squares, Topo 31Z; open
squares, Topo III
. Panel D, the graph
presented in panel C has been expanded to emphasize the points
in which the data of the four different topoisomerases overlap. Closed circles, Topo III; open circles, Topo I; closed squares, Topo 31Z; open squares, Topo
III
. The percentage of the reaction that was present as
fully decatenated Form II molecules was (in panel A) 4.9% (lane 1), 5.7% (lane 2), 6.9% (lane 3), 6.7% (lane 4), 7.4% (lane 5), 4.3% (lane 6),
27.8% (lane 7), 51.6% (lane 8), 97% (lane
9), and 97.7% (lane 10) and (in panel B) 4.2% (lane 1), 9.6% (lane 2), 33.2% (lane 3),
67.5% (lane 4), 92.8% (lane 5), 4.1% (lane
6), 7.0% (lane 7), 12.3% (lane 8), 37.6% (lane 9), and 56.7% (lane
10).
Interestingly,
in contrast to the 15-20-fold stimulation of DNA relaxation
activity compared with that of the truncated enzyme, the addition of
the carboxyl-terminal amino acid residues of Topo I to the truncated
enzyme (Topo 31Z) had only a minimal effect on the resolution of DNA
replication intermediates (Fig. 6B, lanes
2-5). The hybrid enzyme showed, at most, a 2-3-fold
stimulation of decatenation activity when compared with the 604-amino
acid truncation of Topo III (Fig. 6B, lanes
7-10). Topo III is 1-2% as active as the
full-length polypeptide, consistent with the DNA relaxation activity
exhibited by the enzyme.
In contrast to Topo I, however, Topo 31Z was capable of completely resolving plasmid DNA replication intermediates (albeit at a reduced efficiency). This may represent an intrinsic and unique property of the first 605 amino acids of Topo III.
Figure 7: Comparison of the DNA protection patterns generated by Topo 31Z and Topo I. Nuclease P1 protection reactions were performed as described under ``Materials and Methods.'' Reaction products were separated through a 15% polyacrylamide gel in the presence of 50% (w/v) urea. Panel A, reactions contained 2 pmol of Topo III (lane 1) and either no Topo 31Z (lane 2) or 1 pmol (lane 3), 2 pmol (lane 4), or 4 pmol (lane 5) of Topo 31Z. Panel B, reactions contained 0.125 pmol (lane 1), 0.25 pmol (lane 2), 0.5 pmol (lane 3), or 1 pmol (lane 4) of Topo I. The topoisomerase-induced cleavage products generated by each enzyme are indicated by the arrows. The bar indicates the 14-base region surrounding the cleavage site that is protected by Topo III.
The carboxyl-terminal residues of Topo I and Topo III are required for the formation of a stable enzyme-substrate complex, and it has been postulated that this region may constitute a generalized DNA binding domain(13, 14) . Since this domain lies outside the region of homology between the two polypeptides, the possibility that the distinct reactions catalyzed by the two enzymes were the result of the properties of their heterologous carboxyl-terminal domains was examined. This was accomplished by determining the biochemical properties of a chimeric enzyme in which the carboxyl-terminal residues of Topo I were substituted for those of Topo III.
A 604-amino acid truncation of Topo III (Topo
III) had been shown to possess a very low affinity for
single-stranded DNA when compared with the full-length
molecule(14) . The protein sequence homology between Topo I and
Topo III breaks around this point in the amino acid sequence comparison
of the two enzymes; therefore, this region was chosen as the site for
splicing the carboxyl-terminal amino acid residues of Topo I to Topo
III. The hybrid enzyme was overexpressed, purified, and assayed for
supercoiled DNA relaxation activity and its ability to resolve DNA
replication intermediates in vitro.
The addition of the
carboxyl-terminal amino acids of Topo I to Topo III was
able to restore the ability of the enzyme to bind to single-stranded
DNA substrates as well as dramatically stimulate
topoisomerase-catalyzed relaxation of negatively supercoiled DNA. The
hybrid enzyme was approximately 50% as active as either intact Topo I
or Topo III. The biochemical properties of relaxation by the hybrid
enzyme (Topo 31Z) as well as cleavage site specificity were identical
to those of Topo III, suggesting that the first 605 amino acid residues
of Topo III also contribute to the unique characteristics of Topo III.
Since the carboxyl-terminal amino acid residues of Topo I could be fused with another topoisomerase molecule and reconstitute functional topoisomerase activity, this region must constitute a distinct and separable domain of the enzyme. In addition, the properties of this domain appear to be critical in determining the biochemical characteristics of both Topo I and Topo III.
Although the
biochemical characteristics of the relaxation activity of Topo 31Z were
very similar to Topo III, Topo 31Z was very inefficient in catalyzing
the resolution of DNA replication intermediates in vitro. The
addition of the amino acid residues of Topo I stimulated the relaxation
activity of Topo 31Z 15-20-fold relative to Topo III and stimulated the binding of the enzyme to single-stranded DNA
to a similar extent (the equilibrium binding of Topo III
to the identical oligonucleotide has been shown to be
0.5-1% of that of the full-length polypeptide(14) ), but
the decatenation properties of Topo 31Z were enhanced only
2-3-fold relative to the truncated enzyme.
An analysis of
binding specificity of both Topo I and Topo 31Z indicated that these
enzymes bind and protect the entire 45-base oligonucleotide substrate.
This result is distinctly different from the 14-base protection pattern
exhibited by Topo III(22) . Therefore, it is clear that the
substitution of the generalized binding domain of Topo I for that of
Topo III alters the manner in which Topo III binds to its substrate and
alters the catalytic properties of the enzyme. A striking difference
between Topo I and Topo III is that in addition to a preference for
single-stranded substrates, Topo I also has a considerable affinity for
double-stranded DNA()(23) . In contrast, only
single-stranded DNA appears to be an effective substrate for Topo III
binding(11, 14) .
A model for the mechanism of both
decatenation and relaxation, based on the properties of the generalized
binding domains of Topo I and Topo III, data from other laboratories,
and from the known three-dimensional structure of Topo I is presented
in Fig. 8. The mechanism of topoisomerase-induced decatenation
is based on the model by Mondragon and colleagues(24) . In this
model (Fig. 8A), one helix (represented by the circle)
is located in the cavity of the inverted ``U'' structure of
the topoisomerase. The generalized DNA binding domain (represented by
the rectangle) is positioned asymmetrically across the body of
the topoisomerase. This domain is responsible for noncovalent
interactions with the substrate 5` to the topoisomerase cleavage
site(22) . Decatenation occurs when a tyrosine residue in the
active site of the enzyme (triangle) transiently nicks the
single-stranded DNA, creating a ``gate'' that allows the
helix located in the cavity of the enzyme to pass through the nick.
Since the generalized DNA binding domain of Topo III binds
single-stranded DNA, the enzyme is always capable of passing a helix
through the gate and, hence, always capable of decatenating the two
molecules. The generalized DNA binding domain of Topo I, however, has a
significant affinity for double-stranded DNA in addition to its
affinity for single-stranded DNA(23) . If
double-stranded DNA were present in the enzyme active site, the gate
would be blocked by the uncleaved strand of DNA since a type I
topoisomerase can only cleave one strand of the substrate. The same
result would occur if the enzyme bound to a region of a single-stranded
substrate that did not contain a Topo I cleavage site. These binding
properties would result in an enzyme that is inefficient at
decatenation. This would also explain the inefficiency observed in Topo
31Z-catalyzed decatenation since, the majority of the time, the enzyme
would be presented with a substrate that is refractory to decatenation.
Figure 8: Model for DNA topoisomerase I- and topoisomerase III-catalyzed decatenation and relaxation. Panel A, model for topoisomerase III-catalyzed decatenation. The generalized binding domain (rectangle) of Topo III has been positioned upon the known three-dimensional structure of Topo I (inverted ``U'')(24) . Since the two enzymes share extensive protein sequence homology within their first 600 amino acids(10) , it is assumed that they will have a similar three-dimensional structure. Decatenation by Topo III requires that the generalized binding domain hold the substrate to be cleaved across the body of the enzyme (the substrate shown is a gapped molecule, where the DNA in the active site region is single-stranded (single wavy line) and the region outside of the active site is double-stranded). The dotted line indicates where the substrate is held within the binding domain. The double-stranded helix to be unlinked (circle) is held in the cavity present within the body of enzyme. The single-stranded DNA is then transiently cleaved by a tyrosine residue (triangle) present in the active site of the enzyme, with the DNA fragment 3` of the cleavage site held covalently through the tyrosine residue and the DNA fragment 5` of the cleavage site held by the generalized DNA binding domain. The helix is then passed through this transient gate, and the nick is resealed. B, model for Topo I-catalyzed relaxation of negatively supercoiled DNA. Relaxation of negatively supercoiled DNA by Topo I requires that the generalized DNA binding domain of Topo I hold a double-stranded DNA across the body of the enzyme. The generalized binding domain simply feeds the double-stranded molecule into this channel resulting in the local denaturation of the molecule into two single strands (this denaturation step is greatly stimulated by negative superhelicity). Relaxation occurs by the transient cleavage of one of the strands (as described above) followed by the strand passage of the opposite strand (shown in boldface) through the gate. This energy is provided by potential energy store stored in the form of superhelicity within the molecule. Decatenation could not be accomplished even if a helix were present in the cavity of the enzyme since the uncleaved strand of DNA would serve to prevent strand passage. A more detailed description is given under ``Discussion.''
Relaxation of negatively supercoiled DNA (Fig. 8B) is accomplished by the binding of double-stranded DNA to the generalized DNA binding domain of Topo I in a mechanism analogous to the bridging model originally proposed by Cozzarelli and colleagues(7, 26) . In this case, the single-stranded region required for cleavage need only be present locally around the active site tyrosine (triangle) of the enzyme. The three-dimensional structure of Topo I indicates that there is a cleft capable of fitting single strand DNA in the vicinity of the active site of the enzyme(24) . The generalized binding domain may simply feed the double-stranded molecule into this channel resulting in the local denaturation of the molecule into two single strands (negative superhelicity is also required at this step). After strand scission, the torque present in the supercoiled molecule would provide the driving force of strand passage through the gate.
The relaxation
properties of the chimeric enzyme (Topo 31Z) mimic those of Topo III.
This may be explained by the fact that the active site of Topo III
lacks the ability to locally unwind the substrate (this would be
unnecessary since the generalized DNA binding domain of Topo III only
binds and feeds single-stranded DNA into the active site); therefore,
relaxation by Topo 31Z (or by Topo III) requires conditions that
produce stable single-stranded regions within the predominantly
double-stranded substrate. This is consistent with the observation that
both Topo 31Z and Topo III require high temperature and low
Mg concentration to efficiently relax supercoiled
substrates. These conditions both favor the stabilization of
single-stranded DNA within a negatively supercoiled molecule.
Ultimately, the reason for the different catalytic properties of Topo I and Topo III will be resolved by a comparison of the three-dimensional structures of the two enzymes. For example, decatenation of two interlinked molecules requires that a type I topoisomerase interact at a node between a single strand gap or nick and the helix of DNA that must be passed through the transient gate in the single strand. A potent decatenating enzyme, such as Topo III, may contain a structure(s) that promotes the creation of a node and/or stabilizes this intermediate.
The crystal structure of the
amino-terminal 596 residues of Topo I has been determined to
2.2-Å resolution(24) . Unfortunately, this structure does
not contain the carboxyl-terminal substrate binding domain. The
structure of carboxyl-terminal domain of Topo I has been determined
using multidimensional NMR methods(25) ; however, since this
structure was obtained from a purified carboxyl-terminal peptide, it is
unclear how this structure relates to the known crystal structure of
the enzyme. However, crystals have been obtained of the full-length
Topo III polypeptide. ()In addition, the availability of a
catalytically inactive Topo III polypeptide that has the same binding
specificity as the active enzyme (22) should allow the
structural determination of a Topo III-substrate complex.