mAMSA resistant human topoisomerase IIb mutation
G465D has reduced ATP hydrolysis activity
The Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, Framlington Place,
Newcastle upon Tyne, NE2 4HH, UK
Received February 2, 2006; Accepted February 24, 2006
Type II Human DNA Topoisomerases (topos II) play
an essential role in DNA replication and transcription
and are important targets for cancer chemothe-
rapeutic drugs. Topoisomerase II causes transient
double-strand breaks in DNA, forming a gate through
which another double helix is passed, and acts as
a DNA dependent ATPase. Mutations in topoII have
been linked to atypical multi-drug resistance. Both
human Topoisomerase II isoforms, a and b, are tar-
evolution approach to identify mutations conferring
resistance to acridines. Here we report mutation
bG465D, which was selected with mAMSA and
DACA and is cross-resistant to etoposide, ellipticine
and doxorubicin. Resistance to mAMSA appears to
resistance mechanism. G465D lies within the B0
domain in the region that contacts the cleaved gate
helix. There is a 3-fold decrease in ATP affinity and
ATP hydrolysis and an altered requirement for mag-
is decreased for the mutated G465D protein. And we
report for the first time the use of fluorescence aniso-
tropy with intact human topoisomerase II.
DNA topoisomerases are a ubiquitous family of enzymes that
are essential for cellular processes such as replication and
transcription. This role makes them important targets for
both anti-bacterial and anticancer chemotherapeutic drugs.
There are two broad sub-families of topoisomerases, type I
which cleave a single strand, and type II which cleave both
strands of a double helix to transport a second helix through
the break. The type II enzymes (Topo II) show a high degree of
sequence and structural homology, with a common domain
structure. There are three major proteolytic cleavage
sites, termed A, B and C which break the enzyme into four
domains (1,2). These three sites lie between the domains. The
N-terminal domain which contains the ATP hydrolysis cata-
lytic centre lies next to the B0domain, and the A0domain lies
next to this. Together the B0and A0domains make up the
cleavage core catalytic centre, and finally there is the
C-terminal domain bearing a nuclear location signal (3–5)
and many phosphorylation sites (6,7).
Biochemical and structural studies have elucidated a
model for the mechanism of action—firstly a DNA helix is
bound into a semi-circular groove with contacts in the B0and
A0domains. This is termed the gate ‘G’ helix. ATP binding at
the N-terminus leads to dimerization within the N-terminal
domain which can trap a second helix, the ‘T’ helix. A con-
formational cascade is triggered which causes the gate helix to
separate into a double-strand break through which the T-helix
is transported (8,9).
ATP hydrolysis is essential for strand passage activity
of topoII, although DNA cleavage may occur in its absence.
The N-terminus of the enzyme has been shown to bind and
hydrolyse ATP during the reaction cycle in a DNA dependent
manner (10–14). Kinetic studies with yeast topoII have shown
that each dimer binds two ATP molecules and hydrolyses one
molecule rapidly. Pior ADP release is the rate limiting step
before hydrolysis of the second ATP (15–17). DNA transport
occurs after hydrolysis of the first ATP and before hydrolysis
of the second (10). It is thought that ATP binding induces
dimer formation and subsequent conformational changes
which close a clamp-like structure at the N-terminus to trap
the T-segment of DNA, and then induce transport of the
T-helix through the core of the molecule and the cleaved
G-helix (17–19). Binding of just one ATP molecule has
been shown to be sufficient to induce conformational change
across the dimer (20,21).
Crystal structures of the N-terminal domains of GyrB
and yeast topoII have been solved, and these both show
two subdomains. Subdomain 1 contains an ATP-binding
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Nucleic Acids Research, 2006, Vol. 34, No. 51597–1607
pocket and subdomain 2 is termed the transducer domain as it
is thought to contact the core region. Residues in subdomain
2 form a loop that contacts the g-phosphate of the ATP
analogue used. This is thought to be part of a signalling mech-
anism within the molecule which causes a cascade of changes
on ATP binding, leading ultimately to the transport of the
T-helix (22,23). The N-terminal structure of the eukaryotic
topoII from yeast suggests that a pore seen on dimerization
isn’t large enough to accommodate a double helix, and that
dimerization trapping the T-helix will result in torsional strain
that will probably force the T-helix through the gate helix,
and into the core of the enzyme (23). This provides further
evidence of the link between conformational change and
progression through the reaction cycle.
Humans have two distinct topoII isoforms, a and b, which
are located on different chromosomes, are encoded by differ-
ent genes and show different expression patterns within the
cell (24). Both of the human isoforms have been shown to be
targeted by the anticancer drug mAMSA (25).
Human Topoisomerase II is a target for anticancer drugs,
one important class of which is the poisons. These drugs act
through stabilizing an intermediate stage of the reaction,
where the enzyme is complexed with the cleaved gate
helix. These complexes can be processed to double-strand
DNA breaks within the cells which triggers cell death, usually
by apoptosis (26).
Resistance to Topo II drugs is a clinical problem, and
may arise via a variety of mechanisms. Mutations affecting
the way the drug interacts with the enzyme can cause resist-
ance (27). Alternatively reduced enzyme activity gives fewer
functional molecules to target and reduced protein expression
gives the same effect (28–30). The ICRF family of drugs has
been found to interact with the N-terminal domain of topoII
and has been shown to overlap the ATP-binding pocket and
bind two halves of the dimer simultaneously as the molecule
dimerizes on binding of ATP (23,31). As a result ICRF has
been found to preferentially target the ATP bound form of the
enzyme. Mutations in the N-terminal ATPase region have
been shown to confer resistance to ICRFs specifically through
inhibition of the interaction between Topo II and drug (32).
The majority of mutations conferring drug resistance have
been identified in the core catalytic centre responsible for
cleavage. Mutations in human topoIIa have been shown to
confer resistance to mAMSA. Mutations R486K and E571K
showed 100- and 25-fold resistance to mAMSA, respectively.
Interestingly both these mutations showed significant cross-
resistance to etoposide. ATP hydrolysis has been shown to
be effected in some drug resistant mutations (33). Mutation
R450Q was first identified in human leukaemic cell line
CCRF-CEM having conferred resistance to teniposide, and
was subsequently found to give cross-resistance to mAMSA.
This mutation showed altered ATP hydrolysis activity (34,35).
mechanism is through inhibition of the enzyme function as
opposed to inhibition of specific interactions with drug.
The acridines, including mAMSA and DACA, are a family
of drugs used clinically which have been shown to target both
Topo IIa and Topo IIb in vivo (25). mAMSA is established
in the treatment of haematological malignancies and breast
cancers (36) and DACA has entered clinical trials for the
treatment of several cancers including ovarian cancer (37).
The study of point mutations in topoII enzymes can help to
elucidate the mechanism of resistance to topoII targeting
drugs, and a random mutagenesis approach such as the one
used here gives an unbiased selection of mutations that can
cause drug resistance.
Here we report for the first time the characterization of
a mutated human topoIIb G465D, selected independently
for resistance to both mAMSA and DACA. Interestingly res-
istance to mAMSA appears to reduce over time. The mutant is
also cross-resistant to a number of other drugs. DNA binding
of topoII to a 40 bp oligo using fluorescence anisotropy
showed that although the KD was unaffected, binding was
altered for bG465D, and was affected by magnesium. There
is no significant difference between the relaxation activity of
the bG465D mutant compared with the wild-type enzyme
bWT and for decatenation when measured at a single time
point; however whendecatenation was measured over time the
rate was significantly reduced in bG465D. ATP hydrolysis is
reduced 3-fold and magnesium-binding properties measured
in decatentation assays are altered.
MATERIALS AND METHODS
Chemicals and drugs
mAMSA, AMCA and mAMCA were kindly supplied by
Prof. B. C. Baguley, Auckland Cancer Society Research
Center, University of Auckland (Auckland, New Zealand).
Etoposide, Doxorubicin and Ellipticine were purchased
from Sigma. DACA was kindly supplied by Dr P. Charlton.
DNA sequencing was carried out by Lark Technologies Inc.
(Essex, UK). Oligonucleotides were ordered from Invitrogen
Amersham Biosciences (Little Chalfont, Buckinghamshire,
UK) and enzymes from New England Biolabs (Hertfordshire,
UK) or Promega (Southampton, UK). kinetoplast DNA
(kDNA) was purchased from TopoGEN (Columbus, OH).
Random mutagenesis, selection with drugs and
Plasmid YEphTOP2bKLM (38), encoding recombinant
human topoIIb residues 46–1621 fused to the first five residues
of yeast topoII was mutated by exposure to 0.1 M hydroxy-
lamine at 75?C for 40 min, and selected for drug resistance
using methods described previously (33,39). Mutagenized
plasmid was transformed into yeast strain JN394top2-4 to
select for mutations conferring resistance to mAMSA,
mAMCA, AMCA and DACA. Yeast transformants were
then grown in the presence of 74.7 mg/ml mAMSA or
0.5 mg/ml DACA for 96 h at 35?C. Drug resistant yeast trans-
formants were selected by plating onto media plates lacking
uracil and containing drug at the concentrations above, and
grown for 3–5 days at 35?C. DNA from drug resistant colonies
was isolated and the possibility of gross rearrangements or
deletions eliminated by analysis of restriction digestion pat-
terns. Selected plasmids were then retransformed into yeast
to verify that resistance was plasmid borne, and mutations
identified by sequencing both strands.
Owing to the large number of resistant yeast isolated it
was not possible to retransform all plasmids. Plasmids not
1598Nucleic Acids Research, 2006, Vol. 34, No. 5
retransformed were screened for mutation G465D using
restriction fragment length polymorphism (RFLP) analysis.
Mutation G465D is caused by a g to a change at position
1395 and gives an additional HphI site (ggt-gat). A 1 kb region
spanning the region encoding the mutation (base numbers
1200–2200) was amplified by PCR, and then digested with
HphI in NEBuffer 4 (New England Biolabs) at 37?C for at
least 1h. Reaction productswere then separated on a 0.8 or 1%
agarose gel, stained with ethidium bromide and visualized
under UV light. G465D mutation gives an additional band
when compared with wild type.
In vivo characterization
To ensure resistance was due to the point mutation and not
other plasmid changes, a 1674 bp fragment between sites
BamHI and PmaCI containing the mutation was exchanged
with the corresponding fragment of unmutagenized plasmid.
DNA sequencing confirmed the presence of the mutation in
used in all in vivo characterization. Sensitivity to a variety of
drugs was determined by continuous exposure on agar plates
containing drug. Yeast cultures of known optical density were
replica plated, and grown for 3–5 days at 35?C. Fold resistance
was determined from WT and G465D growth at different
concentrations of drug. This method allows rapid analysis
of cross-resistance to many different drugs (27). Resistance
to mAMSA was further quantified by measuring the minimum
lethal concentration (MLC) for bWT and bG465D. Yeast were
exposed to drug in liquid culture for 6 or 24 h, and then grown
on drug free plates to determine the MLC as described pre-
In vitro characterization
Protein purification was either as described previously (2)
or modified as follows: yeast lysis was using Yeastbuster
Protein Extraction Reagent (Novagen), and following elution
from phosphocellulose protein was further purified on a
heparin column. Expression of recombinant protein was in
yeast strain Jel1Dtop1 bearing plasmid YEphTOP2bKLM or
Decatenation and relaxation assays were carried out in
‘relaxation buffer’ (50 mM Tris–HCl, pH7.5, 0.5 mM
EDTA, 1 mM DTT, 100 mM KCl, 30 mg/ml BSA), plus
2 mM ATP, 10 mM MgCl2and either 1 mg of supercoiled
pBR322 plasmid DNA (for relaxation assays) or 400 ng of
kDNA (for decatenation assays). The method was described
previously (2,40). With time-dependent decatenation the
quantified band is expressed as a percentage of total lane
OD to account for background intensities.
Cleavage assays with 40 bp oligonucleotide and a
4.3 kb linearized plasmid were carried out as described
DNA-binding measurements using fluorescence
DNA-binding capacity was determined with purified protein
and a hexachloroflourescein (HEX) labelled 40 bp double-
stranded DNA oligo using fluorescence anisotropy. Meas-
urements were carried out at 20?C using an SLM-Amnico
8100 spectrofluorometer (SLM-Aminco, Urbana, IL). The
excitation wavelength was 530 nm with an excitation slit
width of 8 mm and the emission wavelength was 570 nm
with an emission slit width of 3 mm. A 1 ml fluorescence
cuvette was used with excitation and emission paths each
of 10 mm. Assays were carried out in anisotropy buffer
(50 mM Tris, pH 8, 5% glycerol, 50 mM KCl, 1% Triton
X-100) supplemented with 100 mg/ml acetylated BSA, and
Topo II proteins were matched for buffers and salt concentra-
tion. HEX-labelled oligo (1 mM) was added to the buffer and a
baseline reading taken. Protein was added as described in
Results and the average anisotropy of 12 readings over 99 s
measured for each titration point. MgCl2(10 mM) was added
to the buffer where described. A one-binding site hyperbola
was fitted to data and the BMAX and KD calculated using
GraphPad Prism 4.
ATP hydrolysis activity determination
ATP hydrolysis was determined using purified protein and a
coupled assay linking ATP hydrolysis to NADH oxidation and
a subsequent decrease in absorbance at 340 nm. Assays were
carried out in assay buffer (50 mM Tris, pH 7.4, 66 mg/ml
BSA, 1 mM DTT, 0.5 mM EDTA, 50 mM KCl and 10 mM
MgCl2) using a quartz cuvette. An aliquot of 2 mM PEP,
0.1 mM NADH, 5 U of lactic dehydrogenase, 2.5 U of pyr-
uvate kinase, 10 mg of plasmid DNA and 1 mM ATP (unless
stated otherwise) were mixed and the absorbance at 340 nm
monitored until stable to eliminate the effect of ADP in the
sample. Protein was added at 75 nM, (unless stated otherwise),
the reaction was mixed well and then absorbance at 340 nm
measured for at least 5 min. The linear rate of change of
Abs340 allows the rate of ATP hydrolysis to be calculated
by assuming that 1 mol of NADH corresponds to 1 mol
Selection of human topoisomerase IIb mutations
resistant to acridines
We have used a forced molecular evolution approach to select
and identify mutations in human Topoisomerase IIb that con-
fer resistance to the acridines mAMSA and DACA. Mutation
G465D was selected independently with both mAMSA and
DACA. The plasmid encoding recombinant human Topo IIb
was randomly mutagenized by incubating with 0.1 M
Hydroxylamine for 40 min at 75?C. The library of mutagen-
ized plasmids was then transformed into the temperature sens-
itive yeast strain JN394top2-4: in this strain the yeast topoII
is active at 25?C (the permissive temperature) but inactive at
35?C (the non-permissive temperature), so growth at the non-
permissive temperature is dependent on complementation by a
functional plasmid-borne topoII. The transformed yeast were
selected for drug resistance by exposure to drug in liquid
culture at either 74.7 mg/ml mAMSA or 0.5 mg/ml DACA,
for 96 h at the non-permissive temperature. Transformants
were further selected by plating onto Ura-plates containing
mAMSA or DACA at the levels stated above and grown
for 3–5 days at the non-permissive temperature. DNA from
drug resistant colonies was isolated and subjected to restric-
tion digest with HindIII to eliminate any plasmids that had
undergone gross rearrangements or deletions. A selection of
Nucleic Acids Research, 2006, Vol. 34, No. 51599
plasmids was retransformed into yeast to verify that the drug
resistance was plasmid borne, and the DNA was sequenced
to identify the mutation. Using this approach mutation G465D
was selected and identified using this approach a total of three
times, twice with mAMSA and once with DACA. A g to a
change at position 1395 causes the glycine to aspartic acid
change. The plasmids that had not been retransformed were
screened for the presence of the G to D mutation at codon
465 using RFLP analysis as this mutation leads to a gain of an
HphI site (ggtgg–ggtga), and G465D was found to have been
selected once more with mAMSA.
Plasmids (500) were selected for ability to grow on plates
containing mAMSA and 3 were sequenced—2 of these were
found to contain a base change g–a at position 1395 which
confers mutation G465D. More than 1000 plasmids were
selected for ability to grow on plates containing DACA, 23
were sequenced and 1 of these was found to contain the base
change g–a and so also conferred mutation G465D. Plasmids
(36) selected with mAMSA, and 240 selected with DACA that
were able to complement yeast topoII were screened by RFLP
analysis to see if they also contained mutation G465D. One
moreG465Dmutation fromthe mAMSAscreenwas identified
giving three in total identified with mAMSA and one with
Mutation G465D confers resistance to mAMSA
Topoisomerase mediated drug resistance can occur through
decreased expression of the enzyme, perhaps through a
promoter defect, giving fewer molecules to target. To verify
that the point mutation identified, G465D, was responsible for
the drug resistant phenotype, and not mutations elsewhere in
the plasmid, fragment exchange was performed on the plas-
mid. A 1674 bp region containing the mutation was excised
and exchanged for the corresponding segment from an
unmutagenized vector. The plasmid was then transformed
into JN394top2-4. The level of mAMSA drug resistance
was quantified using the MLC method (39). JN394top2-4
transformed with either bWT or bG465D was grown in the
presence of different concentrations of mAMSA for 6 or
24 h, then the amount of growth relative to a t0plate quantified
for both yeast strains. The bG465D transformed yeast showed
?10-fold resistance to mAMSA at 6 h with MLC values of 1
and 10 mg/ml for bWT and bG465D, respectively (Figure 1A).
This difference was statistically significant as determined by
a two-tailed paired t-test (P < 0.05). Interestingly after 24 h
of exposure only 2-fold resistance to mAMSA was seen with
MLC values of ?2 and 5 mg/ml seen for bWT and bG465D,
respectively. This difference wasn’t found to be statistically
significant. (Figure 1B). Exposure of bG465D to mAMSA for
3–5 days on drug plates showed very low levels of resistance,
with growth very similar to wild type (data not shown).
G465D confers cross-resistance to many different drugs
Mutations givingtopoisomerase IIdrugresistanceoftenconfer
cross-resistance to other drugs, so we compared the drug
resistance phenotypes of yeast transformed with wild-type
or mutant plasmid. The two yeast transformants were
grown on YPDA plates supplemented with drugs of various
concentrations, after 3–5 days the level of growth was
compared. The drugs tested were from a variety of functional
classes, these being acridines mAMSA, AMCA, mAMCA,
DACA, the epipodophyllotoxin Etoposide, the anthracycline
Doxorubicin and Ellipticine. Mutation bG465D showed
resistance to all except mAMSA. The mutant also showed a
2-fold resistance to AMCA, 5-fold resistance to mAMCA and
10- to 20-fold resistance to DACA, a 4-fold resistance to
etoposide, 10-fold resistance to ellipticine and 2-fold resist-
ance to doxorubicin (data not shown).
Topo IIb proteins were purified to 95% homogeneity as
described previously (2). To try and determine the mechan-
ism for drug resistance the DNA cleavage properties of
Figure 1. MLCmeasuringsurvivalofyeasttransformedwithplasmidencoded
WTb and bG465D on growing with mAMSA for 6 h (A) or 24 h (B) at 35?C.
Experiments were repeated three times.
1600 Nucleic Acids Research, 2006, Vol. 34, No. 5
bG465D vs bWT were assayed. The proteins were incubated
with a 40 bp32P-labelled linear DNA substrate and the reac-
tion stopped by addition of SDS to denature the protein. Pro-
teinase K removes the protein and allows the DNA fragments
to be analysed by PAGE. Cleavage reactions were performed
with either magnesium ions, the natural divalent cation for
topoII, or calcium ions which are commonly used to enhance
the cleavage reaction (41). Topoisomerase poisons are also
known to enhance cleavage by stabilizing the cleavable com-
plex, and cleavage assays were done in the presence and
absence of mAMSA. Relative cleavage with wild-type and
mutant enzyme in several experiments was calculated by
quantifying the amount of end-labelled cleaved product by
(Fujifilm, Tokyo, Japan). The amount of 40 bp oligo substrate
cleaved into the two products by the wild-type protein bWT in
the presence of magnesium and mAMSA was quantified and
this was taken as 100% and cleavage under other conditions
calculated relative to this. Cleavage of wild-type protein with
magnesium was stimulated 12-fold on addition of mAMSA
(100%). Cleavage in the presence of calcium ions increased
5-fold (42 ± 7.7%) compared with magnesium alone (7.8 ±
2.4%), and was highest in the presence of calcium and
mAMSA (376.4± 97.6%).
magnesium was 28.2 ± 10.3 and 82.2 ± 29% in the absence
and presence of mAMSA, respectively. Calcium enhanced
bG465D cleavage with values of 63.5 ± 51.9 and 250.6 ±
65% in the absence and presence of mAMSA, respectively.
The averagevalues for triplicate
cleavage experiments were lower than for the triplicate
bWT experiments. However the SDs were rather large, so
when a student t-test was carried out the differences were
Cleavage of the 40 bp DNA substrate can also be stabilized
by etoposide. No significant difference in the ability of
bWT and bG465D to cleave in the presence of etoposide
was observed, with cleavage at 100 and 92 ± 8.9%,
The cleavage patterns with a
pBR322 fragment for bWT and bG465D enzyme were com-
pared. The cleavage pattern with this substrate was similar to
that of bWT, the same major cleavage products were seen but
some bands were of lower intensity (Figure 2).
32P-end-labelled 4.3 kb
DNA binding of bWT and bG465D to a HEX-labelled 40 bp
oligo was measured using fluorescence anisotropy, in the pres-
ence and absence of 10 mM MgCl2. Fluorescence anisotropy
was calculated relative to the baseline reading. Without
supplemented magnesium the maximum anisotropy seen for
bWT and bG465D was 0.1998 ± 0.002474 and 0.1535 ±
0.004687, respectively, a significant difference as determined
by a two-tailed unpaired t-test (P > 0.05; Figure 3A). When
the reaction is supplemented with 10 mM MgCl2there is an
increase in bWT anisotropy to 0.2701 ± 0.02286, a significant
difference (P > 0.05). bG465D also increases significantly
(P < 0.0001) to 0.2180 ± 0.01203 in the presence of magnes-
ium (Figure 3B and C). However the KD of binding is not
significantly different between bWT and bG465D with values
of 9.429 ± 0.6234 and 11.34 ± 1.694, respectively.
Strand passage activity
Strand passage activities of purified proteins were assayed
with relaxation and decatenation assays. A minimum of
three independent experiments were carried out for each
assay, for a single time point (30 min), and the wild-type
activity taken as 100%. The relaxation activity of bG465D
was not significantly different from bWT at 104.8 ± 8.3 and
100%, respectively. While the decatenation activity appeared
slightly reduced at 60.3 ± 18.2%, this was not significantly
different from bWT (100%) in a t-test.
The rate of strand passage activity was also investigated
by assaying the decatenation of 400 ng of kDNA by 1 U of
enzyme over time. While bWT reached maximum decatena-
tion after 20 min, bG465D begins to reach saturation after
120 min (Figure 4). The initial rate of change in intensity per
minute (expressed as the change in the % lane OD per minute)
for each enzyme was determined with regression analysis
and found to be 4.913 ± 0.6413 and 0.4011 ± 0.1232 for
bWT and bG465D, respectively, an ?12-fold decrease in
initial decatenation rate.
Figure 2. Cleavage with mAMSA and a 4.3 kb linear substrate. Protein
(1 pmol) was incubated with 100 mg/ml Etoposide (lanes 2–3) or 50 mg/ml
1 and 4 are with no protein, lanes 2 and 5 are with WTb, and lanes 3 and 6 are
Nucleic Acids Research, 2006, Vol. 34, No. 5 1601
An increased ATP requirement and narrower MgCl2
optima is seen with G465D
The ATP requirements of the mutant enzyme were measured
using decatenation assays at a variety of ATP concentrations
(Figure 5A). Interestingly the bG465D mutant showed a
3-fold increase in ATP requirement compared with the
bWT. The bWT reaches maximum decatenation at ?50 mM
mM ATP, at which point bG465D shows only 40% decatena-
tion. bG465D reaches 100% decatenation at 150 mM ATP, a
3-fold increase. This is confirmed with Kmvalues; 19 mM for
bWT and 62 mM for bG465D, an increase of 3.26-fold.
The response of the mutant to MgCl2,an essential cofactor,
was also measured by increasing the MgCl2concentrations in
decatenation assays (Figure 5B). While the bWT and bG465D
both reached maximum activity at 10 mM MgCl2, the bG465D
showed a much narrower optima. The wild-type enzyme
showed continuing maximum decatenation until 20 mM
MgCl2, then ?80% at 30 mM, 45–50% at 40 mM and 40%
at 50 mM. This correlates with previous results (40). The
bG465D mutant gave 80% decatenation at 20 mM, 30% at
30 mM and ?20% at 40–50 mM MgCl2. The Kmfor both
proteins is similar; however, as there is no data between
0 and 10 mM MgCl2, where activity is already maximal,
this cannot be measured accurately.
Mutation G465D gives reduced ATP hydrolysis activity
As the mutant has an increased ATP requirement for decat-
enation and lies towards the N-terminal ATPase domain ATP
hydrolysis activity assays as described in Materials and
Methods were carried out to determine if this alteration was
because of impaired ATP hydrolysis activity. ATP hydrolysis
assays were carried out in 10 mM MgCl2, shown to be optimal
for both proteins. Assays with increasing concentrations of
enzyme showed clearly that the bG465D protein had severely
impaired ATP hydrolysis activity as compared with wild type.
The slope determined from linear regression was 0.7477 ±
0.1744 and 0.0387 ± 0.01193 for bWT and bG465D, respect-
ively (Figure 6A). An analysis of the Vmaxof both wild-type
and mutant enzymes showed that the wild-type maximum
was 3.68-fold higher than that of bG465D at 31.99 nmol
ATP per second and 8.702 nmol ATP per second, respectively
(Figure 6B), which is in agreement with the 3-fold increase in
ATP requirement to perform decatenation seen with the mut-
ant. The ATP hydrolysis activity of human topoisomerase IIb
Figure 3. Fluorescence anisotropy Bmaxvalues for bWT and bG465D. Protein
(300 nM) and HEX-labelled 40 bp oligo (1 mM) were used per experiment.
MgCl2was added to 10 mM where appropriate. Protein was added in 5 ml
Anisotropy of bWT and bG465D apoproteins. (B) Anisotropy of bWT and
bG465D in the presence of 10 mM MgCl2. (C) Comparison of maximum
anisotropy for bWT and bG465D in the presence and absence of MgCl2.
Figure 4. Decatenationovertime.Enzyme(1U)wasusedtodecatenate400ng
of kDNA at 37?C for increasing lengths of time as shown. Bands were quanti-
fied using TINA densitometry software and then expressed as a % of the total
representing 1 SD from the mean.
1602 Nucleic Acids Research, 2006, Vol. 34, No. 5
is unstable, and diminishes quickly after protein purification.
To ensure that the ATPase activities of both proteins were
optimal, the proteins were purified in the same week, flash
frozen in liquid nitrogen and stored at ?80?C and experiments
done the following week.
Using a forced molecular evolution approach, selecting with
mAMSA and DACA, drug resistant mutation G465D has been
identified in topoisomerase IIb on four occasions with two
selection agents. This mutation also confers resistance to sev-
eral other drugs of different classes including AMCA,
mAMCA, Etoposide, Ellipticine and Doxorubicin.
The resistance to DACA is very strong at 10- to 20-fold
compared with wild-type enzyme. DACA is unlike the other
acridines in that its exact mechanism of action is not
well understood, with evidence suggesting it acts as a dual
topoisomerase I and II poison, so caution must be
exercised in interpreting the drug resistance data from the
yeast organism. DACA may not be exclusively targeting
Topo II (42,43).
An atypical drug resistance phenotype such as that given by
bG465D, where resistance is conferred to a range of structur-
ally diverse drugs, implies that the mechanism of resistance is
through a reduction in enzyme function giving fewer func-
tional molecules to target as opposed to a decrease in specific
interaction with drugs. Mutations of this type have been
reported previously in the B0domain (26).
Figure 6. (A)ATPhydrolysisactivitywithincreasingprotein.Reactioncarried
A single dataset is shown. Data were plotted in GraphPad Prism 4. (B) ATP
hydrolysis with increasing ATP. Reaction was carried out in reaction buffer
containing 10 mM MgCl2with 9 mg of DNA and 75 nM protein in each case.
ATP was increased as described. Maximum determined by GraphPad Prism 4.
A single dataset is shown.
Figure 5. (A) Dependence of decatenation activity on ATP concentration.
Protein was used at a level sufficient to decatenate 80% of DNA substrate,
using 400 ng of kDNA. Maximum decatenation for each protein is taken as
100% with other values expressed as a percentage of this. ATP dependent
activity is shown for bWT (squares) and bG465D (triangles). Results are
the average of three experiments and error bars represent 1 SD from the mean.
to decatenate 80% of DNA substrate, 400 ng of kDNA was used per reaction.
Maximum decatenation for each protein is taken as 100% with other values
expressed as a percentage of this. Magnesium dependent activity is shown for
bWT (squares) and bG465D (triangles). Results are the average of two experi-
ments and error bars represent the SD from the mean.
Nucleic Acids Research, 2006, Vol. 34, No. 51603
The resistance to mAMSA, the selection agent in three
of the four G465D mutations identified, was measured at dif-
ferent time points. The MLC for strains expressing either
bG465D or bWT was measured at 6 and 24 h and continuous
growth on drug plates was measured after 3–5 days. A clear
pattern emerged where the drug resistance decreased over
time, from a 10-fold resistance after 6 h exposure, to 2-fold
resistance after 24 h exposure, and then after 3–5 days
bG465D showed no resistance. These data imply that,
while a defect in the enzyme is likely, this defect does not
permanently disable any aspect of enzyme function and
merely ‘slows’ the turnover.
Indeed, the yeast carrying the mutant plasmid grows at
approximately half the rate of yeast with plasmid encoding
wild-type Topo IIb (data not shown). The selection method
used ensures that any mutants identified will be functional—if
they weren’t then the yeast would be unable to grow at the
non-permissive temperature, thus the slower growth of the
bG465D yeast implies that while it is functional, it is less
functional than wild type. If there is less functional topoII
target it would give a drug resistance phenotype.
The relaxation shows no significant difference to wild
type which is unexpected as an enzyme with lower general
function would be expected to have a slower reaction cycle. It
is however possible that the experimental set up may be mask-
ing a slight impairment of activity. The bG465D decatenation
for a single time point showed just 60.3% wild-type activity
and subsequent decatenation versus time experiments showed
an initial 12-fold rate reduction. Interestingly the bG465D
displayed a narrower magnesium optima compared with
bWT. In decatenation experiments the MgCl2concentration
was 10 mM, optimal for both bWT and bG465D proteins, and
perhaps more importantly the ATP levels were saturating,
enough perhaps to compensate for the enzyme’s natural defi-
ciency in ATP hydrolysis. ATP dependence assays showed
maximum activity at 150 and 50 mM ATP for bG465D
and bWT, respectively, and in decatenation assays ATP
was at 2 mM.
The reason for this reduced decatenation rate is unclear. It
is clear that the ATP requirements for bG465D differ mark-
edly from bWT. Decatenation assays with varying concentra-
tions of ATP showed a 3-fold decrease in ATP affinity with
bG465D, and a corresponding 3-fold decrease in ATP hydro-
lysis activity with this mutant. This is particularly interesting
as, while the mutation does lie towards the N-terminal ATPase
domain, it is actually located in the B0domain, close to where
the G-helix is predicted to bind (8,44).
Mutations in this region have been reported for human
topoIIa and yeast topoII. The topoIIa mutation R450Q (equi-
valent to bK466), lies adjacent to the mutation identified here
(34,35). This mutation gives a 2.5-fold resistance to mAMSA
and was first identified in the drug resistant leukaemic cell line
CEM/VM-1, which was found to have a 2- to 8-fold decrease
in ATP hydrolysis activity. In the case of yeast topoII
residue G437S, equivalent to bG464, a loss of enzyme stabil-
ity, reducing the number of functional target molecules, has
been reported previously as a reason for drug resistance (45).
The activity profile differed to that of bG465D and aR450Q
in that G437S showed drug hypersensitivity and no alteration
in ATP requirement and degradation was very rapid. Extra-
polating between species can be problematic and has been
described previously (33). Reduced stability is not seen
with bG465D, so this is unlikely to be the mechanism of
resistance in this case.
The primary sequence of topoIIb places G465 adjacent to
the transducer domain of the N-terminal ATPase domain,
which is thought to function by transmitting the ‘information’
of ATP hydrolysis from the N-terminus to the cleavage core
through a series of conformational changes. Experiments with
WTa have shown that a two-residue insert at position 408,
equivalent to bE425 (46), and a deletion stretching from 350
to 407 (47) can disrupt communication between domains. In
these cases inter-domainal communication was completely
abolished, and it is conceivable that a residue contacting
this domain may have a communication role. Additional splice
variants of human topoIIa have been identified with differ-
ently sized inserts in the transducer region of the ATPase
domain, at positions K321, Q355 and A401 (48). It is possible
that these alternative forms will have differences in the trans-
duction of conformational changes which could account for
differences in activity in vivo.
The proximity of the residue to the transducer domain also
makes it possible that this inhibits signal transduction through
conformational change. The yeast core crystal structure, on
which all of the topoII models are based, lacks any data for the
region linking the core and ATPase domains as this was too
disordered, so it is unclear how these two interact. It is likely
however that the ATP hydrolysis activity of the mutant is
reduced through inhibition of signal transduction through
the transducer domain to the N-terminal region.
The position of G465 on the model based on the yeast
structure, plus previous footprinting experiments suggest
that glycine 465 lies very close to the area binding the gate
helix. Lysines in yeast topoII have been identified that are
protected on DNA binding, and one such residue identified
was Lys 438. The equivalent residue in WTb is K466 which is
adjacent to G465 (8,44). Modelling the G465D mutation onto
the yeast core structure using SwissProt shows that while
the residue lies in the GyrB homology domain, and would
therefore be expected to be involved in ATP hydrolysis, in
eukaryotes this is actually located in the B0domain, on the top
surface of the semi-circular groove where the gate helix is
thought to bind (Figure 7A–D) (8,44). The mutation of residue
465 from a glycine to an aspartic acid doesn’t appear to
massively change the 3D structure of the enzyme, which is
expected as our selection procedure would not select a non-
functional mutant.Analysis of this region shows that the muta-
tion gives rise to some localized hydrogen bonding changes
that could potentially alter the conformation of the enzyme
(Figure 7E and F). The presence of the negatively charged
aspartic acid creates an additional hydrogen bond to the back-
bone and could create more resistance to conformational
It is possible that the replacement of a glycine with an
aspartic acid, and hence the addition of a negative charge,
could alter the charge interactions involved in binding of
the gate helix. Fluorescence anisotropy experiments with a
40 bp oligo showed a decreased BMAX value for bG465D,
suggesting that the protein is binding in a slightly different
conformation giving a consequent change in tumbling rate.
However there was no significant difference in KD for the two
proteins implying that binding affinity was not effected. This,
1604 Nucleic Acids Research, 2006, Vol. 34, No. 5
Figure 7. Model ofhuman topoIIb monomerbased on the yeast core structure.The effect of the mutationwas modelledusingSwiss-PdbViewer 3.7, the figures
from the side, the same orientation as (B) and (D).
Nucleic Acids Research, 2006, Vol. 34, No. 51605
along with the fact that the oligo used was not physiological,
suggests that it is unlikely that impaired DNA binding is
responsible for the decrease in decatenation seen with
The narrower optimum for MgCl2is interesting and could
perhaps be explained by considering its role in the topoi-
somerase II mechanism. Mg2+is thought to be utilized at
both the ATP-binding domain and in the cleavage core. In
the nucleotide-binding pocket Mg2+has been shown to contact
all of the phosphate groups as well as an invariant Asn residue
(23,49). In the structure of the N-terminal region of human
topoIIa it is also shown to hydrogen bond with a water
molecule that is also forming contacts with Glu87, a residue
found previously to be a general base ideally positioned to
promote nucleophilic attack on the g-phosphate (50). It is
possible that the altered Mg2+optima seen could be linked
to an impairment in the nucleotide-binding pocket, whereby
Glu87 is unable to promote ATP hydrolysis.
bG465 lies within the core region, where Mg2+is thought
to be involved in polarizing the active site tyrosine and sta-
bilizing the cleavable complex intermediate, and is therefore
co-ordinated near to the site ofgate helix binding. There isalso
evidence for a structural role for Mg2+in this region (27).
In this region it is thought that Mg2+is co-ordinated to a
catalytic triad of aspartic acid residues and the active site
tyrosine, allowing nucleophilic attack on the phosphate of
DNA (40,51). The mutation of glycine 465 to aspartic acid
introduces a residue that may also co-ordinate Mg2+. The
cleavage site is likely to have a higher affinity for Mg2+
than the D465 region, which would explain why at lower
Mg2+concentrations both proteins are equally active, but at
higher Mg2+levels D465 may also co-ordinate magnesium in
protein could either interfere in the phosphoryltransfer reac-
tion directly, or it could impair the movement of domains
necessary for enzyme turnover.
The structure of human topoIIa shows that when AMPPNP
(an ATP analogue) is bound the transducer domain assumes a
packed conformation in which a catalytic lysine residue
protrudes into the nucleotide-binding pocket and contacts
the g-phosphate. When ADP is bound however the transducer
domain is rotated outward 10?, retracting the lysine from the
pocket, in a conformation thought to be necessary for Pi
release (49). It is thought that the Pi release event and removal
of the so-called ‘switch lysine’ from the nucleotide-binding
pocket may be coupled simultaneously with the separation of
the G-segment in the cleavage core (52). Our data are con-
sistent with this hypothesis, and it is probable that mutation of
glycine 465 to aspartic acid impedes the communication
between domains mediated by transducer region, giving a
reduced ATP hydrolysis rate and slower enzyme turnover.
It is known that the transduction of signal through con-
formational change is necessary for co-ordination of core
and N-terminal domains to give functional enzyme. Here
we report a mutation which lies in the core domain, and yet
gives a 3-fold reduction in activity at the N-terminal domain
implying that transduction of signal has been affected. This
mutation confers a resistance to drug that diminishes with
time, suggesting that rather than being fatally impeded the
enzyme is merely slowed down, a mechanism of resistance
which to our knowledge is previously unreported.
We would like to acknowledge Rosalind Turnbull for preli-
minary experiments, and Gary Watters and Pauline Heslop for
work done on the RFLP analysis, Dr Ian Cowell for help with
figures, Prof. Bernard Connolly for advice regarding fluores-
cence anisotropy experiments and Prof. Harry Gilbert for
advice with ATP hydrolysis assays. C.L. was funded by
Cancer Research, UK; K.G. was funded by the Institute for
Cell and Molecular Biosciences, The University of Newcastle
Upon Tyne. Funding to pay the Open Access publication
charges for this article was provided by Knowledge House.
Conflict of interest statement. None declared.
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