(Received for publication, July 18, 1994; and in revised form, June 14, 1995)
From the
The interactions of the Escherichia coli endonuclease
UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II)
(complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with
DNA than cis-DDP and gives monoadducts with a longer lifetime
(8 h 20 min chelation t compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [H]DNA,
the filter binding assay was associated with the analysis of the
nucleoprotein complexes to characterize the UvrAB recognition of the
platinum adducts and to demonstrate the occurrence of platinum-mediated
DNA-protein cross-linking. First, it is shown that the UvrAB proteins
recognize the complex 1 mono- and diadducts with a higher affinity than
those of cis-DDP. Fifteen times more cis-DDP adducts
per plasmid are required than complex 1 adducts, to lead to similar
UvrAB binding. However, the UvrAB proteins recognize monoadducts and
diadducts of each complex with a similar affinity. Second, it is shown
that UvrB is the protein involved in the nucleoprotein complexes formed
from mono- and diadducts of complex 1 and cis-DDP. This
protein is also partly cross-linked to DNA with a similar efficiency by
monoadducts derived from complex 1 and cis-DDP. However, as
UvrB has a greater affinity for the DNA adducts of complex 1 than for
those of cis-DDP, more UvrB-platinum-DNA cross-links are
formed with complex 1 than with cis-DDP. This study, using a
bacterial repair system as a model, points to a possible strategy for
making new cytotoxic platinum complexes for mammalian cells.
cis-Diamminedichloroplatinum(II) (cis-DDP) ()is one of the most widely used anticancer chemotherapeutic
agents. It is thought that this compound exerts its cytotoxic effects
through damage to DNA by activating a programmed cell
death(1, 2) . Several experimental arguments suggest
that the cis-DDP biological properties might result from the
specific recognition, by high affinity proteins, of the DNA structural
motif induced by intrastrand chelation. Such proteins have been
isolated and characterized(3, 4, 5) . It was
shown that HMG1 and several other cellular proteins like SSRP1 which
contain the same sequence motif, called HMG box, were implied
in such recognition. HMG1 or HMG2 proteins are implied in transcription
processes(6, 7) . This led the authors to suggest that
intrastrand diadducts could induce a DNA structural motif which would
mimic the natural binding site of such proteins. According to this
hypothesis, cis-DDP activity would result from two nonmutually
exclusive events due to the binding of HMG box containing
proteins to the intrastrand diadducts: (i) the trapping of these
proteins causing cell death by perturbating the transcription of
critical genes, (ii) the inhibition of the binding of repair proteins
preventing the excision of lesions. All these observations suggest that
platinum derivatives able to form intrastrand diadducts should elicit
pharmacological properties.
Searching for new platinum derivatives,
Hollis et al.(8, 9) synthesized cis-[Pt(NH)
(Am)Cl]
complexes, where Am is an heterocyclic or aromatic amine ligand
like pyridine, pyrimidine, purine, or aniline. These compounds form
only monoadducts with DNA. Among them, some elicited antitumor
properties, although the monoadducts were not recognized by the
cellular SSRP1 protein(6) . Since these monoadducts were shown
to inhibit DNA replication, it was proposed that their cytotoxic effect
was due to the inability of the DNA repair systems to eliminate the
lethal lesions. These results suggest, that depending on the nature of
the nonleaving ligands, monoadducts could exhibit antitumor properties.
Recently, in a cooperative program with our laboratories,
Rhône-Poulenc Rorer synthesized platinum complexes
characterized by bulky and hydrophobic substituted ethylenediamine
ligands(10, 49) . These ligands were chosen in order
to increase the affinity of the platinum compounds for the DNA major
groove and also to decrease the chelation rate of the platinum
monoadducts. Preliminary studies revealed that the rate of monoadducts
formation from these complexes was higher than that observed for cis-DDP and that their chelation was indeed very slow. Several
of these compounds, like the norbornyl derivative of Fig. 1(complex 1), were found to exhibit strong antitumor
properties(10, 49) . Furthermore, these compounds were
able to overcome cis-DDP resistance in L1210 cells. These
observations suggest that their mechanism of action might be different
from that of cis-DDP and might involve a biological processing
of the monoadducts. Platinum mono and diadducts are repaired in
prokaryotic and eukaryotic
cells(2, 11, 12, 13, 14, 15, 16, 17, 18) .
Recent studies on trans-1,2-diaminocyclohexanedichloroplatinum(II), which is
also characterized by a bulky nonexchangeable ligand, revealed that
monoadducts were more efficiently repaired than diadducts by UvrABC
excinuclease(14) . The recognition of monoadducts still bearing
a labile ligand, chloro or aqua, could lead to its substitution by a
protein nucleophilic group. Therefore, such monoadducts might become
lethal lesions because of their ability to cross-link repair proteins
to DNA. Such DNA-protein cross-linking was not observed with (DACH)
dichloroplatinum complexes; however, the authors did not study the
interaction of UvrABC excinuclease with monoadducts which still had a
labile aqua or chloro ligand.
Figure 1: Structures of the platinum complexes.
In order to test this hypothesis, the UvrAB recognition (19, 20) of complex 1 and cis-DDP DNA adducts was studied using the filter binding assay. The determination of the rate constants of the first platination and chelation steps, together with the use of a technique which rapidly eliminates unbound platinum allowed us to discriminate between the recognition of chloro monoadducts and that of the final diadducts. The cross-linking of UvrAB proteins to DNA via chloroplatinum adducts was determined. The results for complex 1 and cis-DDP are discussed.
The reaction was stopped by increasing the NaCl concentration
to 0.5 M and cooling at 0 °C. Samples were dialyzed twice versus high-salt TEN buffer (TEN plus 0.5 M NaCl) at
0 °C. The samples were precipitated with 0.8 volume of isopropanol,
suspended at 1 mg/ml in high-salt TEN. Samples were then dialyzed versus two changes of NaClO 10 mM, pH
5.5, for 2 h at 0 °C in order to remove chloride.
After aliquots
were removed for determination of platinum content by AAS and of DNA
concentration, the samples were divided into 200-µl aliquots and
either left at 4 °C (controls) or incubated at 37 °C. The
amount of monoadduct present at each time point was determined by
adding 20 µl of 110 mM [C]thiourea
(specific radioactivity of 5.23 µCi/µmol) and incubating for 10
min at 37 °C. The reaction was stopped by adding 2 ml of cold 5%
trichloroacetic acid and cooling to 4 °C. In order to reduce the
background, samples were pelleted, the supernatant was removed by
aspiration, and the pellet was redissolved in 0.2 ml of 0.1 N NaOH. After reprecipitation of the DNA with cold 5%
trichloroacetic acid, the samples were collected by filtration on a
glass fiber filter for further analysis.
The formation
of stable nucleoprotein complexes between UvrAB and platinated DNA was
measured by trapping the native protein-DNA complexes onto
nitrocellulose filters as described(22) . The 140-µl
reaction mixture consisted of 40 mM potassium
morpholinopropane sulfonate buffer at pH 7.5, 85 mM KCl, 1
mM dithiothreitol, 15 mM MgSO, 2 mM ATP (binding buffer), 200 fmol (each protein) of UvrA and UvrB,
and 14 fmol of [
H]DNA treated or not treated with
platinum compounds. After 10-min incubation at 37 °C, the reaction
was stopped by addition of 5 ml of cold 2
SSC (0.3 M NaCl, 0.03 M trisodium citrate). After 5 min in ice in
order to eliminate nonspecific binding, the nucleoprotein complexes
were collected by trapping onto a BA85 nitrocellulose filter.
All the data were analyzed using nonlinear regression (GraphPAD program, San Diego, CA).
In the text, the results of the binding assay (Fig. 4A and 5A) are expressed as t values which correspond to the incubation time between DNA and the platinum complex required to obtain half the fraction of DNA retained on the filter at infinite time.
Figure 4:
UvrAB binding to DNA treated with the
platinum complexes to form mainly monoadducts. DNA was incubated with
the platinum complexes at 37 °C in NaClO (10
mM, pH 5.5) as described under ``Materials and
Methods.'' At the times indicated, unbound platinum complexes were
removed by two spun G10 chromatographies, and platinated DNA was
incubated with UvrA and UvrB proteins. Then, the filter binding assay
was performed (see ``Materials and Methods''). Results are
the mean of three independent experiments and are expressed with the
standard error of the mean. A, UvrAB binding to platinated DNA as a
function of the incubation time between DNA and the platinum complexes.
, complex 1;
; cis-DDP. The results were analyzed
by nonlinear regression using GraphPad system (San Diego, CA). The best
fits were obtained with exponential and sigmoid curves respectively for
complex 1 (R
: 0.98) and cis-DDP (R
: 0.999). B, comparison of the relative UvrAB
recognition efficiencies for DNA-monoadducts of complex 1 and cis-DDP.
The apparent number of proteins bound per plasmid DNA (
u)
was determined from the fitted curves of A according to , where
is the filter retention efficiency (see
text), and u is the average number of proteins bound to DNA.
The amount of platinum per plasmid was computed from the fitted curves
of Fig. 2. The filter retention efficiency is similar for both
platinum complexes, since binding experiments were performed in the
same conditions. The apparent average number of proteins bound per
plasmid is expressed as a function of the platinum amount per plasmid.
Linear regressions give
u = 0.3
platinum-adducts per plasmid (r = 0.993) and
u = 0.02
platinum-adducts per plasmid (r = 0.998), respectively, for complex 1 and cis-DDP. Solid line, complex 1; dotted line, cis-DDP.
Figure 2:
Quantification of platinum bound to DNA as
a function of incubation time. H-Labeled plasmid DNA (6.9
µM) was treated with platinum complexes at a concentration
of 1.7 µM (r
= 0.25) at 37
°C in 10 mM NaClO
at pH 5.5. Quantification
was performed using AAS.
, complex 1;
, cis-DDP.
Results were fitted with sigmoid curves. The R
values were 0.997 and 0.995, respectively, for complex 1 and cis-DDP.
Since platinum derivatives interact
preferentially with guanine that can be considered to be distributed at
random, the location of platinum adducts results in a Poisson
distribution. The UvrAB proteins binding reflects the amount of binding
of platinum to DNA. Therefore, the number of proteins bound
specifically to platinum adducts is also described by a Poisson
distribution. The apparent average number of proteins bound to DNA
(u) can be expressed as
follows,
where is the filter binding efficiency which is considered
as the probability that a protein complexed to DNA at a particular
site, is held on the filter, u is the average number of
proteins bound to DNA, and f is the fraction of DNA retained
on the filter(23, 24) .
The UvrAB
binding and the isolation of DNA-protein complexes were performed using
a method described previously (25, 26) after slight
modifications. This method was as follows: 6.7 nM of H-labeled pSP65, treated as above, was incubated with UvrA
and UvrB proteins, both 94 nM, at 37 °C in 80 or 140
µl of binding buffer containing 20% glycerol. After 10 min of
incubation at 37 °C, samples were loaded onto an AcA22 gel
exclusion column (1.0
13 cm) equilibrated in the same binding
buffer with 300 mM KCl at 25 °C. The column was run at 4
ml/h, and 700-µl fractions were collected. The DNA content of each
fraction was quantified by scintillation counting of 50-µl
aliquots. For protein determination, aliquots corresponding to the peak
fraction and equivalent either to 90 or 120 ng of DNA were analyzed by
SDS-PAGE and silver staining.
Figure 3:
Kinetics of monoadducts to diadducts
conversion. Salmon sperm DNA was treated with platinum derivatives in
order to obtain a r of 0.015 (see ``Materials
and Methods''). Monoadducts to diadducts conversion was performed
by incubating platinated DNA in NaClO
10 mM, pH
5.5, at 37 °C. At the times indicated, aliquots were taken and the
r
of monoadducts was determined by incorporation of
[
C]thiourea. The percentage of monoadducts was
determined by the ratio of the monoadduct r
on the
total platinum r
.
, complex 1;
, cis-DDP.
In order to determine the UvrAB binding efficiency on
DNA diadducts, we allowed the monoadducts free of unbound platinum
compounds to undergo the chelation step before addition of the UvrAB
proteins. At various platination times, samples were filtered on G10
columns and incubated for 20 h at 37 °C. This length of incubation
was shown to produce more than 80 and 99% diadducts, respectively, from
complex 1 and cis-DDP (data extrapolated from Fig. 3). The filter binding assay yielded the following t of incubation to obtain half the maximum protein binding: 1.9
± 0.44 and 32.5 ± 1.4 min, respectively, for complex 1 and cis-DDP (Fig. 5A). The apparent number
of proteins bound to DNA as a function of platinum per plasmid was
analyzed. Linear regression gave u = 0.3
platinum adducts per plasmid and 0.03
platinum adducts per
plasmid, respectively, for complex 1 and cis-DDP. About
10 times more cis-DDP diadducts than complex 1 diadducts are needed to obtain a similar apparent average number of
proteins bound to DNA (Fig. 5B). Since the experimental
conditions were identical to those described for monoadducts (similar
), UvrAB proteins recognize complex 1 diadducts with a
better efficiency than those formed from cis-DDP.
Figure 5:
UvrAB binding to DNA-platinum diadducts.
DNA was treated as described in the legend to Fig. 4, but after
filtration on G10 column, the samples were incubated 20 h at 37 °C
in NaClO 10 mM, pH 5.5, to allow the conversion of
monoadducts to diadducts before incubation with UvrAB proteins. Then,
the filter binding assay was performed. All the data were analyzed as
described in the legend to Fig. 4. A, UvrAB binding to
DNA platinum diadducts as a function of the ANA platination time. The
best fits were obtained with exponential and sigmoid curves,
respectively, for complex 1 (R
: 0.993) and cis-DDP (R
: 0.997).
, complex 1;
, cis-DDP. B, comparison of the relative UvrAB
recognition efficiencies for DNA-diadducts of complex 1 and cis-DDP. The apparent average number of proteins and the
amount of platinum bound to DNA were computed as described in the
legend to Fig. 4B. Linear regressions gave
u = 0.3
platinum adducts per plasmid (r = 0.996) and
u = 0.03
platinum
adducts per plasmid (r = 0.999), respectively, for
complex 1 and cis-DDP. Solid line, complex 1; dotted line, cis-DDP.
Figure 6:
Binding of UvrA and UvrB proteins to DNA
containing mainly either mono- or diadducts of complex 1 or cis-DDP. Reaction mixtures in 80 µl of binding buffer
containing 94 nM UvrA and UvrB, 6.7 nMH-labeled pSP65 bearing either 30 mono- or diadducts
of complex 1 per plasmid, or 40 mono- or diadducts of cis-DDP
per plasmid were loaded onto a gel exclusion column. 700-µl
fractions were collected and their DNA content quantified by
scintillation counting. Peak fraction aliquots equivalent to 90 ng of
DNA were analyzed by SDS-PAGE and silver staining. St, UvrA
and UvrB proteins used as standards. In this experiment, the UvrA
standard migrated as a doublet resulting from oxidation of its
sulfhydryl groups(21) . Lane a, non platinated DNA; lanes b and d, DNA containing, respectively, 30 and
40 monoadducts of complex 1 and cis-DDP per plasmid; lanes
c and e, DNA containing respectively 30 and 40 diadducts
of complex 1 and cis-DDP per plasmid. ★, limit of the
stacking gel. Nonplatinated DNA enters into the stacking gel whereas
platinated DNA does not. In lane c, one can observe a weak
smear. Such a smear was only observed with DNA containing complex 1
diadducts and might result from the degradation of a small amount of
platinated DNA during the 20-h incubation at 37 °C in
NaClO
.
Figure 7:
Cross-linking between UvrAB proteins and
platinated DNA. DNA samples containing increasing amounts of
monoadducts or diadducts were prepared as described in the legends of Fig. 4and Fig. 5. The platinated DNA was incubated with
UvrAB proteins as described under ``Materials and Methods.''
After a 10-min incubation with UvrAB proteins, samples (140 µl)
were divided into two parts. One part was used as a control for the
filter binding assay (data not shown). To the second part, SDS and EDTA
(0.2% SDS, 50 mM EDTA final concentration) were added, and the
samples were incubated at 37 °C for 30 min. After dilution by 5 ml
of warmed 2 SSC buffer and an incubation at 37 °C for 5
min, the nucleoprotein complexes resistant to SDS/EDTA treatment were
collected onto nitrocellulose filter. A, determination of
SDS/EDTA-resistant nucleoprotein complexes as a function of the DNA
platination time between DNA and the platinum complexes. UvrAB addition
after DNA platination:
, complex 1;
, cis-DDP.
UvrAB addition after 20-h incubation at 37 °C following platination
of DNA:
, complex 1;
, cis-DDP. B,
comparison of the amounts of protein cross-linked to DNA adducts of
complex 1 and cis-DDP. The apparent average number of proteins
and the amount of platinum per plasmid were determined as described in
the legend of Fig. 4B. UvrAB addition after DNA
platination: solid line, complex 1; dotted line,
cis-DDP. UvrAB addition after 20-h incubation at 37 °C
following platination: dashed line, complex 1; no SDS/EDTA
resistant nucleoprotein complexes were observed with cis-DDP
in this range of adduct ratio.
Figure 8:
Dissociation of the noncross-linked
UvrB-platinum-DNA complexes by SDS/EDTA treatment. Reaction mixtures in
140 µl of binding buffer containing 94 nM of UvrA and UvrB
(each protein), 6.7 nMH-labeled pSP65 bearing,
respectively, 30 and 40 monoadducts of complex 1, and cis-DDP
per plasmid were incubated or not with 0.2% SDS, 50 mM EDTA at
37 °C for 30 min. The non-SDS/EDTA-treated samples were loaded onto
a gel exclusion column. The SDS/EDTA-treated samples were dialyzed
twice against 2
SSC containing 1 mM dithiothreitol and
20% glycerol for 1 h at 37 °C in order to eliminate SDS and loaded
onto a gel exclusion column equilibrated in a modified binding buffer
containing 50 mM KCl, 50 mM EDTA, 50 µM dithiothreitol, and deprived of ATP. 700-µl fractions were
collected. Aliquots (90 ng of DNA) were analyzed by SDS-PAGE and silver
staining. ★, limit of the stacking gel. Lanes a and c, non-SDS/EDTA-treated samples containing, respectively, DNA
monoadducts of complex 1 and cis-DDP; lanes b and d, SDS/EDTA-treated samples containing, respectively, DNA
monoadducts of complex 1 and cis-DDP.
Figure 9: Demonstration of the formation of cross-linked UvrB-platinum-DNA complexes using a thiourea treatment. Aliquots (120 ng of DNA) corresponding to SDS/EDTA-treated samples of Fig. 8(lanes b and d) were incubated for 24 h at 37 °C in the presence (+) or in the absence(-) of 1 M thiourea, 0.3 M Tris-HCl, pH 7.5. In the absence of thiourea, the samples were incubated in 0.3 M Tris-HCl, pH 7.5. Then samples were analyzed by SDS-PAGE and silver staining. St, UvrA and UvrB standards; lanes a and b, DNA containing, respectively, 30 and 40 monoadducts of complex 1 and cis-DDP; lane c, DNA containing 30 diadducts of complex 1. ★, limit of the stacking gel. As already observed in the Fig. 6, platinated DNA does not enter the stacking gel until it was treated with thiourea treatment which dissociates platinum adducts from DNA. It has to be noted that the thiourea treatment does not interfere with silver staining. Furthermore, a silver-stained smear is observed in all the wells even in the unloaded wells. Such a smear results from the heavy silver staining of the gel.
The racemic complex, cis-[N-2-amino N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II) with exo configuration (complex 1) bears a bulky hydrophobic norbornyl substituent on the ethylenediamine ligand. It was designed to have a higher affinity than cis-DDP for DNA major groove and also to give monoadducts with longer lifetimes.
Two questions were addressed in this work: (i) do the UvrA and/or UvrB proteins of the Escherichia coli repair system exhibit different recognition efficiencies for the complex 1 and cis-DDP DNA adducts and for the mono- and diadducts of each complex and (ii) does complex 1 yield long-living monoadducts and are such adducts able to cross-link the UvrA and/or UvrB proteins to DNA?
For the chelation step, the
monoadducts of complex 1 are more slowly converted into
diadducts than those of cis-DDP, with respective t of
8 h 20 ± 20 min and 2 h 40 ± 30 min (Fig. 3). A
decrease in the chelation rate was already reported for
PtCl(DACH) compared with PtCl
(en) and cis-DDP (t: respectively 4 h 24, 1 h 50 min, and 2 h
20 min, (14) ). It is noteworthy that the trans-1,2-diaminocyclohexane ligand is hydrophobic, but less
bulky than the norbornyl ethylenediammine ligand.
The UvrABC endonuclease is known to recognize a variety of DNA
damages (19) . This broad spectrum led the authors to postulate
that the UvrABC endonuclease rather recognizes the DNA structural
deformation induced by the various adducts than the characteristic
features of each adduct. Despite the absence of structural data on the
mono- and diadducts of complex 1 with DNA, it is likely that
they are similar to those of the corresponding cis-DDP
adducts. For the latter, it is known that the square planar
coordination of platinum(II) is the major factor controlling the
stereochemistry of the adducts and that it induces DNA bending for the
intrastrand diadducts (31) but not for the
monoadducts(32, 33, 34) . Therefore one
expected different UvrAB recognition of the mono- and diadducts for
both complex 1 and cis-DDP. The UvrAB proteins
recognize a variety of adducts even the O-methylguanine ones which cause little distortion
of the DNA helix(35) . Our results showing a similar
recognition of both the mono- and diadducts of complex 1 and cis-DDP suggest that simple structural determinants do not, by
themselves, control the UvrAB recognition. Our UvrAB results for a
population of lesions located randomly on a supercoiled DNA can be
compared with the UvrABC repair results of defined adducts reported by
Page et al.(14) . They found a better UvrABC repair
efficiency for the [G]Pt(dien) or [G]PtCl(en) and
[G]PtCl(DACH) monoadducts trapped with dithiothreitol than
for the [GG] diadducts of cis-PtCl
(DACH). Visse et al.(36) concluded from footprinting experiments that the
recognition complex involves binding of the proteins to the convex side
of the bend formed by the platinum GG-chelate. Clearly, this does not
explain the similar recognition of the mono- and diadducts of complex 1 and of cis-DDP. The norbornyl ethylenediamine ligand
is bulkier than the two ammine ones, and molecular mechanics modeling
of the mono- and diadducts shows that both remain in the major groove
of the straight and bent structures (not shown). The more efficient
binding of UvrB to both mono- and diadducts of complex 1 suggests that hydrophobic interactions might be favorable to
recognition in the major groove area. Several results suggest that the
UvrABC, or actually UvrAB recognition efficiency, is not directly
related to the UvrABC incision efficiency. The latter depends upon the
torsional constraint of the damaged DNA, whereas the former does not (22, 37) . Using supercoiled DNA, Beck et al.(12) showed that UvrABC incised cis- and trans-DDP-treated DNA with the same efficiency, whereas using
linear DNA, they found that only the one treated with cis-DDP
was incised. UvrABC incision efficiency is also dependent upon the
sequence of the damaged DNA strand. It has been observed that the
UvrABC incision efficiency of a DNA treated with acetylaminofluorene
varied with the DNA sequence, whereas recognition did not(38) .
All these results suggest that the UvrAB recognition and UvrC incision
steps might obey different binding criteria. The recognition would work
for bent DNA structures as well as for any type of adducts,
particularly with bulky hydrophobic groups, whereas incision would be
more sensitive to the DNA sequence and the torsional
constraint(39) .
In this work, bacterial UvrAB proteins were used for their ability to recognize DNA lesions induced by platinum monoadducts and diadducts. In mammalian cells, the specificity of the recognition and incision steps as well as the nature of the proteins are less characterized. Several proteins involved in repair or in transcription processes have been isolated for their ability to recognize platinum DNA adducts(2, 47, 48) . Analogues of cis-DDP able to react quickly with DNA, to give long-lived monoadducts able to cross-link repair proteins may be good candidates to overcome the repair-mediated cis-DDP resistance(2) .
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