From the
Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadron, 45071
Orléans Cedex 02, France,
Génoscope-Centre National de Séquençage, 2, rue Gaston
Crémieux CP 5706, 91057 Evry Cedex, France
Received for publication, February 7, 2003
, and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recently, studies showed that the DNA mismatch repair (MMR) pathway
contributes to the cytotoxicity elicited by cisplatin. In eukaryotes, MMR has
been involved as an upstream component of a cisplatin-induced apoptotic
pathway mediated by the tumor suppressor p73 or by the retinoblastoma tumor
suppressor (21,
22). Importantly, a
relationship between the loss of a functional MMR pathway and cellular
resistance to cisplatin cytotoxicity has been established in vitro
and in vivo (23,
24,
25,
26). As cisplatin resistance
is a major reason for treatment failure, it is therefore of great interest to
understand how MMR-dependent molecular mechanisms participate in the
modulation of cellular sensitivity to the drug. Several models have been
proposed to explain the possible biochemical link between the MMR pathway and
cisplatin cytotoxicity (reviewed in Refs.
26,
27,
28,
29) but no precise mechanism
has yet been defined. An important molecular determinant of such mechanisms
was the identification of MMR proteins implicated in cisplatin cytotoxicity.
Early experiments in Escherichia coli have shown that loss of MMR
activity because of mutations in mutS or mutL was correlated
with cisplatin resistance
(30). In eukaryotes, mismatch
repair defects because of loss of either human MSH2 or MSH6 subunits of
hMutS or the human MLH1 or PMS2 subunits of the hMutL
complex
result in cisplatin resistance
(23,
24,
26). Thus MutS or MutL and
their respective eukaryotic counterparts hMutS
or hMutL
are MMR
proteins implicated in cisplatin cytotoxicity.
Because the primary role of MutS and hMutS appears to be recognition
of mispaired bases and small base insertion mismatches
(31,
32,
33) and as recognition of
cisplatin-damage DNA is a necessary step in all proposed models, the first
mechanistically important question concerns both the capability of MMR
proteins to sense cisplatin-damaged DNA and the identification of the
cisplatin lesions specifically recognized. A quantitative study reported that
hMutS
displayed weak affinity for 1,2-d(GpG) cisplatin intrastrand
cross-links (34) but in
contrast a qualitative study reported that this protein exhibits a strong
preference for cisplatin compound lesions formed when a thymine is
misincorporated opposite the major cisplatin adduct
(35). This protein was also
found to have a reduced affinity for a G/T mismatch in the context of a
1,2-d(GpG) adduct (13)
suggesting that compound lesions were not involved in MMR activity
(36). hMSH2 alone also
displayed weak affinity for the major intrastrand cross-link
(37). E. coli MutS
was shown to recognize DNA globally modified by cisplatin
(38) but the precise nature of
the bound adducts is not yet known. Therefore, a sensitive assay is needed to
gain insight into the nature of the suspected cytotoxic lesions recognized by
MMR binding activities. In particular, no extensive interaction study has been
carried out with cisplatin compound lesions, defined here as a cisplatin
lesion in one strand and a mismatch in the other.
In the present study, we investigated the binding properties of E. coli MutS protein with various platinated DNA substrates containing a single centrally located cisplatin adduct to define the precise nature of the bound adducts. Experimental conditions were used in which sensitivity was high because of the absence of carrier DNA and to low nonspecific background binding. Within the sequence context studied here, the 1,2-d(GpG) adduct is the only bifunctional cisplatin lesion that is recognized albeit with a very low relative affinity. In contrast, MutS recognized with higher specificity an extended array of cisplatin compound lesions that are likely to be formed in vivo based on cisplatin mutation spectra. Surface plasmon resonance was employed for the first time to study MutS binding kinetics to several platinated DNA substrates; the weak binding of MutS to the 1,2-d(GpG) intrastrand cross-link is essentially dependent on a fast rate of dissociation. Taken together, our interaction data with MutS and different platinated DNA substrates suggest that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are the most probable critical lesions for MMR-mediated cisplatin cytotoxicity.
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EXPERIMENTAL PROCEDURES |
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Protein PurificationHexameric histidine-tagged MutS protein
was prepared as previously described
(39). The host strain was BL21
(DE3) (Novagen) and the expression vector pMQ382 was kindly provided
by Dr. M. G. Marinus. The strain transformed with pMQ382 was grown at 37
°C with shaking to an A600 of 0.6. The culture was
then shifted to 25 °C and induced with
isopropyl-1-thio-
-D-galactopyranoside at a final
concentration of 50 µM for 3 h. After centrifugation, the cells
were resuspended in a sonication buffer (20 mM Tris-HCl, pH 7.6,
500 mM NaCl, 20 mM imidazole, 5 mM
-mercaptoethanol, 10% glycerol) supplemented with protease inhibitor
mixture (Sigma), frozen at -20 °C, and then sonicated. All purification
steps were performed at 4 °C. The lysate obtained after centrifugation of
the extract was mixed with a nickel affinity resin (Qiagen) and MutS was
batch-eluted with elution buffer containing 100 mM imidazole. After
overnight dialysis MutS was stored at -80 °C in a buffer containing 350
mM NaCl, 20 mM Tris-HCl, pH 7.6, 0.1 mM EDTA,
1 mM dithiothreitol, and 50% glycerol. The concentration of MutS
protein was determined using the Bradford reagent (Pierce) with bovine serum
albumin as standard and the purity was estimated as >95% using Coomassie
staining of a sodium dodecyl sulfate-acrylamide gel. The concentration was
1.52 mg/ml.
Preparation of the glutathione S-transferase-MutY fusion protein
was carried out as previously described
(41). The host strain was BL21
(DE3) and the expression vector pGEX-MutY was kindly provided by Dr.
S. Yonei. The strain transformed with pGEX-MutY was grown at 25 °C with
shaking to an A600 of 0.6. Expression of the glutathione
S-transferase-MutY fusion protein was induced by the addition of 50
µM isopropyl-1-thio-
-D-galactopyranoside, and
growth was continued overnight at 25 °C. After centrifugation, the cells
were resuspended in phosphate-buffered saline supplemented with protease
inhibitor mixture, frozen at -20 °C, and then sonicated. After
centrifugation the supernatant was applied to a glutathione-Sepharose 4B
column (Amersham Biosciences) at 4 °C. The bound protein was eluted with
15 mM glutathione in 50 mM Tris-HCl, pH 8. After
dialysis, the glutathione S-transferase-MutY fusion protein was
cleaved in phosphate-buffered saline by thrombin protease by overnight
incubation at 4 °C and the final concentration was 0.5 mg/ml.
Platination and DNA PurificationThe sequences of the
oligonucleotides are shown in Fig.
1. Single-stranded oligonucleotides including those with a
3'-biotin label were purified on a 24% denaturating polyacrylamide gel.
The preparation and purification of oligonucleotides containing single
1,2-d(GpG), d(ApG), or d(GpCpG) cisplatin intrastrand cross-links and the
synthesis of the cisplatin interstrand cross-link was as described
(42). The single-stranded
purine-rich oligonucleotide 5'-AGGAGTAGAGATCGAGAGAGTAAG-3' was
5'-end labeled with T4 polynucleotide kinase and
[-32P]ATP. Radiolabeled heteroduplexes GG/CT and G*G*/CT
were prepared by mixing the 32P-labeled oligonucleotide with a 1.5
molar excess of complementary purine-rich oligonucleotide containing a GG
platination site without or with a cisplatin residue in annealing buffer (60
mM KCl, 5 mM Tris-HCl, pH 7.5, 0.1 mM EDTA),
heating at 75 °C for 5 min and slowly cooling at 4 °C. Radiolabeled
duplexes GG/CT and G*G*/CT were then purified on non-denaturating 10%
polyacrylamide gels. Unlabeled duplexes for competition experiments were
annealed by combining equimolar amounts of the two complementary strands at
concentrations of 13.5 µM in annealing buffer at 75
°C followed by cooling to 4 °C.
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Electrophoretic Mobility Shift Competition AssaysMutS (21 nM) was incubated in binding buffer (100 mM NaCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, 80 µg/ml acetylated bovine serum albumin, and 2% glycerol) with mixtures of 32P-labeled duplex GG/CT (2 nM) and particular unlabeled duplex competitors at varying concentrations as indicated in 20 µl. Reactions were allowed to reach equilibrium by incubation at 4 °C for 20 min. Gel loading buffer was added, and the reaction mixture was loaded on a 6% native polyacrylamide gel (37.5:1, acrylamide:bisacrylamide (w/w); 45 mM Tris borate, 0.5 mM EDTA, pH 8.3). Gels were electrophoresed for 1 h at 4 °C at 13 V/cm, dried, and exposed to an Amersham Biosciences PhosphorImager screen overnight. The fraction bound (radioactivity of the bound complex/total radioactivity) was determined by ImageQuant 5.1 software (Amersham Biosciences). Data analysis were carried out as described (43). We determined the concentration of various competitors necessary to decrease by 2-fold the amount of DNA shifted in the absence of competitor by linear regression of plots of the fraction of DNA bound in the absence of competitor divided by the fraction of DNA bound in the presence of competitor as a function of the competitor concentration.
Direct Binding ExperimentsVarying concentrations of MutS (0 to 100 nM) were used to titrate 32P-labeled duplex GG/CT or G*G*/CT (2 nM) in binding conditions as described above with the exception that no competitor DNA was present. The subsequent steps were the same as described under "Electrophoretic Mobility Shift Competition Assay." The fraction bound was calculated and plotted as a function of the protein concentration. Data were least-squares fitted to an equation for a simple two-state binding process (44) using Origin 6.0 software (Microcal, Northampton, MA).
Surface Plasmon Resonance (SPR) MeasurementsSPR measurements were performed on a BIAcore 2000 at 15 °C using a streptavidincoated chip and duplex DNA containing a 3'-biotin on the nonadducted purine-rich strand. 24-Mer oligonucleotides used for SPR were the same as those presented in Fig. 1 with the exception that the purine-rich strand oligonucleotides have a 3'-biotin. Various biotinylated duplexes were prepared by mixing biotinylated purine-rich strand with a 3-fold molar excess of pyrimidine-rich strand in annealing buffer at 75 °C followed by cooling to 4 °C. Platinated and unplatinated biotinylated DNA duplexes were bound to the streptavidin-coated SA sensor chip by injecting 10 µl of 750 mM NaCl, 10 mM HEPES-KOH, pH 7.4, 3 mM EDTA, 0.005% surfactant P20, and 1.7 µM biotinylated duplexes GG/CC, G*G*/CC, GG/CT, G*G*/CT, GG/TC, or G*G*/TC. MutS protein was injected at a flow rate of 20 µl/min in running buffer (100 mM NaCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, 0.0005% Tween 20, and 2% glycerol). Two washes of 10 µl of running buffer containing 0.04% SDS were used to regenerate the surface after each injection of MutS. Binding to each duplex was assayed at five different concentrations of MutS (01000 µM). To obtain kinetic parameters, late association phases and early dissociation phases of the resulting sensorgrams were fit to the Langmuir binding model using BIAevaluation software (version 2.0). The ratio of bound MutS per duplex DNA substrate was deduced from the stoichiometry formula (MutS response x DNA Mr)/(DNA response x MutS Mr), where MutS response is the maximal response (in RU) obtained at the plateau level, DNA response (in RU) corresponds to the plateau value for a DNA substrate, and DNA and MutS Mr are molecular weights of the DNA substrate and MutS, respectively.
To check the chemical stability of bound platinum residue within biotinylated duplexes, the platinated pyrimidine-rich strand was 5'-32P-end labeled followed by hybridization with matched or mismatched complementary biotinylated strand. Labeled duplexes were then incubated in 100 mM NaCl, 5 mM MgCl2, 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA at 25 °C for 24 h and loaded on a 24% denaturating polyacrylamide gel. After migration no product corresponding to unplatinated pyrimidine-rich strand was detected indicating that the platinum residue was not displaced from the strand.
MutY Adenine Glycosylase AssayRadiolabeled double-stranded 24-mer oligonucleotides GG/CC, GG/AC, and G*G*/AC were prepared and purified as described above (with the purine-rich strand 5'-32P-end-labeled). Reactions were as previously described with minor modifications (41). Briefly, duplexes (40 fmol) were incubated with MutY (130 ng) at room temperature in 10 µl of reaction buffer containing 20 mM Tris-HCl, pH 7.5, 50 µg/ml bovine serum albumin, and 10 mM EDTA followed by the addition of 2 µl of 2.5 M NaOH and heating at 95 °C for 5 min at the times indicated. The denaturated samples were electrophoresed on a 24% denaturating polyacrylamide gel followed by quantification by phosphorimaging.
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RESULTS |
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MutS Recognition of Cisplatin Intrastrand Cross-links When a Mismatched
Thymine Is Opposite One Platinated Guanine hMutS was found
to recognize more efficiently a d(GpG) adduct when it is part of two compound
lesions formed after misincorporation of a thymine opposite either 3'-
or 5'-platinated guanine
(35). Because these substrates
have not been studied for binding with MutS, we first investigated the
recognition by MutS of the duplex G*G*/CT containing a 1,2-d(GpG) cisplatin
intrastrand cross-link with a thymine opposite the 3'-modified guanine.
As shown in Fig. 3, MutS binds
strongly to the mismatched G*G*/CT substrate. The determined
Kd value of 7 ± 1 nM shows that
the presence of the adduct stimulates the binding activity of the protein by
about 3 times as compared with the unplatinated G/T substrate
(Kd = 19 ± 3 nM, above). As
shown in Fig. 4 and
Table II, this difference in
binding was also confirmed in competition experiments. Next, we tested
recognition of another compound lesion in which a thymine residue is now
located opposite the 5'-platinated guanine of the 1,2-d(GpG) adduct. The
duplex G*G*/TC competes less efficiently than GG/TC
(Fig. 4); the presence of the
adduct decreases the affinity of the protein with a factor of 4-fold
(Table II). These data indicate
that according to the position of the mismatched thymine, the cisplatin adduct
can stimulate or can impair MutS recognition. Interestingly, this effect has
also been observed with hMutS
(35) suggesting that both
bacterial and human proteins exhibit similar binding properties with cisplatin
compound lesions.
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In contrast to the stimulation by the adduct to binding of MutS with the duplex G*G*/CT, a DNA substrate containing a cisplatin 1,2-d(ApG) intrastrand cross-link with a T opposite the platinated guanine competes with the same order of magnitude as the corresponding unplatinated heteroduplex (Fig. 4, Table II). Thus, the 1,2-d(ApG) intrastrand cross-link can be recognized because of the presence of the misincorporated thymine opposite the adducted guanine.
MutS Recognizes an Extensive Set of Cisplatin 1,2-Intrastrand Cross-link/Base MismatchesResults on cisplatin mutagenicity in both prokaryotic and eukaryotic cells indicate that several different compound lesions can be produced and that adenine or guanine rather than thymine are preferentially mispaired opposite 1,2-intrastrand adducts (14, 15, 16, 17). To identify the compound lesions specifically bound by a MMR binding activity, we analyzed interaction of MutS with platinated duplexes containing all other possible mispaired bases opposite the 5'- or the 3'-platinated base of the 1,2-d(GpG) or -d(ApG) cross-links. Again, the abilities of various mismatched DNA substrates with or without a 1,2-intrastrand cross-link to compete with radiolabeled duplex GG/CT were compared by competition experiments; the relative binding affinities for MutS are reported in Table II. The trend in affinity for mismatches is G/T > G/G > G/A for unplatinated duplexes in the GG series and G/T > G/G >A/C > A/A > G/A for unplatinated duplexes in the AG series. This order of affinity qualitatively agrees with that reported previously, and the specific recognition of these mismatches by MutS is consistent with their repair both in vitro and in vivo (46, 47). Among nine platinated duplexes of the GG and AG series with G/G, G/A, A/A, and A/C mismatches, seven are specifically bound by MutS (Table II). This result illustrates the capacity of MutS to bind DNA containing a large variety of cisplatin compound lesions. Mutagenesis studies have shown that d(GpG) and d(ApG) adducts primarily induced, respectively, G to T and A to T transversions and less frequently G to C and A to C transversions, with a preponderance located at the 5'-modified base in prokaryotes (14). Our finding that MutS recognizes specifically the 1,2-d(GpG) adduct when the complementary strand contains an adenine or a guanine opposite the 5'-platinated guanine and the d(ApG) adduct with a mispaired adenine opposite the 5'-platinated adenine strengthens the likelihood of a role of such compound lesions in mechanisms linking cisplatin cytotoxicity and MMR.
Among the seven platinated duplexes with mispaired bases opposite either modified base that we have found to act as specific competitors, five are more effective than the corresponding unadducted heteroduplexes. The increase in affinity occurs with the major cisplatin adduct for all G/A and G/G mispairs tested and is independent of the mispair location (opposite 3'- or 5'-platinated guanine). As compared with the mismatched duplexes, the presence of 1,2-d(GpG) adduct increased the specificity of MutS by 5-, 3-, and 2-fold for the duplexes G*G*/CG, G*G*/AC, and G*G*/GC, respectively. This effect also occurs with 1,2-d(ApG) adduct in the case of the duplex A*G*/TA. Inversely, the presence of this adduct can decrease the relative ability of a mismatched substrate to compete, as observed with the competitor A*G*/TG. An even more dramatic effect was observed with the duplexes A*G*/CC and A*G*/GC as the presence of the adduct abolishes MutS recognition of A/C and A/G mismatches, respectively. These results indicate that the nature of the 1,2-intrastrand adduct can play a differential role in mismatch recognition of cisplatin compound lesions.
SPR Analysis of MutS Binding to Platinated DNA SubstratesWe used SPR to examine the kinetics of binding of MutS to oligonucleotide duplexes containing either a 1,2-d(GpG) intrastrand cross-link or a 1,2-d(GpG) adduct with a mismatched thymine opposite either the 5'- or 3'-platinated guanine.
For binding of platinated and unplatinated 24-base pair duplexes to the Biacore streptavidin chip, a biotin residue was added to the 3' end of the unadducted purine-rich strand. Because platinum has affinity for sulfur donors (48), we showed that the stability of the 1,2-d(GpG) adduct within a duplex was unaffected by the presence of a sulfur-containing biotin residue (see "Experimental Procedures"). After immobilizing the appropriate biotinylated duplexes to separate flow cells, MutS was injected over the surfaces. As shown in Fig. 5A, the maximum level of binding of MutS to the heteroduplex GG/CT is much higher as compared with that of the perfectly paired duplex, as expected for a mismatch binding protein. As deduced at saturation binding obtained at a higher protein concentration (RUmax = 650, data not shown), MutS as a dimer was found to bind to the heteroduplex in a 1:1 manner, in agreement with previous studies (40, 49). Adding a cisplatin 1,2-intrastrand adduct to the GG/CC homoduplex results in an increase of the plateau level. A similar increase occurred when the mismatched thymine was opposite the 3'-platinated guanine. Examination of the association and dissociation phases shows that the kinetic parameters responsible for the affinity increase are different for homoduplex and heteroduplex: with the homoduplex, this increase relates to variations of the dissociation rate, whereas for the heteroduplex it is the association step that is changed. In contrast, when the mismatched thymine is opposite the 5'-modified guanine, the plateau level of the platinated heteroduplex is lower (Fig. 5B). In this case, MutS dissociation is more rapid as compared with the corresponding unplatinated mismatched duplex GG/TC. The SPR data are in qualitative agreement with results obtained from competition experiments.
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The association and dissociation rate constants and the relative affinities of MutS for the different DNA substrates were determined by injecting increasing concentrations of MutS through the cells (data not shown). Most of the kinetograms were fitted with a 1:1 Langmuir model. The association rate for the MutS-G*G*/CT complex was not determined because of a complex biphasic type of interaction; a fit of the dissociation phase using a single exponential decay model provided the dissociation rate constant for this platinated substrate. Summary of data are reported in Fig. 5C. Our thermodynamic parameters compare well with those previously obtained with G/T-containing DNA substrates (50). The dissociation rate of MutS from the platinated homoduplex is 2-fold lower as compared with the corresponding unplatinated duplex and the affinity decrease for duplex G*G*/TC is related to a 4.8-fold increase in MutS dissociation as compared with duplex GG/TC. Thus, these data confirmed that the variations in the dissociation phase can explain the differences in MutS affinity for duplexes G*G*/CC and G*G*/TC as compared with their corresponding unplatinated duplexes.
Compound Lesions with Adenine Opposite Either Platinated Guanine of the Major Cisplatin Intrastrand Cross-link Are Not Substrates for the Bacterial DNA Glycosylase MutYAlthough MutS recognizes specifically a 1,2-d(GpG) cisplatin cross-link when an adenine is opposite either platinated guanine, it is possible that in vivo, these lesions are primarily substrates for MutY, a DNA glycosylase responsible for the first step of base excision repair of adenine misincorporated opposite guanine or opposite the damaged guanine, 8-oxoguanine (51). Due to its glycosylase activity, it is difficult to study the binding of MutY with DNA substrates containing a G/A mismatch. Therefore, we compared the glycosylase activity of MutY with the same 24-mer duplexes as above containing a mispaired adenine opposite unplatinated or platinated guanine in a 1,2-d(GpG) cross-link. In the experimental conditions used here, apurinic sites generated by the removal of mispaired adenines by MutY were chemically converted to strand breaks. The resulting fragmented products were then analyzed on a denaturating gel. As shown in Fig. 6A, the 24-mer double-stranded oligonucleotide GG/AC bearing a mismatched adenine yields a product comigrating with a 12-mer oligonucleotide thus showing the glycosylase activity of MutY. No cleavage was detected with the corresponding matched duplex GG/CC. Incubation of MutY with a DNA substrate containing a mispaired adenine opposite the 5'-platinated guanine of 1,2-d(GpG) cross-link did not result in cleavage. The same result was obtained with duplex G*G*/CA (data not shown). This loss of activity could result from either a lack of duplex recognition by MutY or an inhibition of the glycosylase activity by cisplatin. To test the former hypothesis, we compared the efficiency of competitor duplexes GG/CC or G*G*/AC in inhibiting the cleavage reaction of the G/A mispair-containing duplex (Fig. 6B). We found that duplexes G*G*/AC inhibited the cleavage reaction but with the same efficiency as the matched duplex GG/CC. The same result was obtained with duplexes G*G*/CA (data not shown). Thus the major cisplatin intrastrand cross-link abolishes the specific recognition of a G/A mispair by MutY. Although competition between MutS and MutY does occur for the repair of G/A mispairs, our data show that it no longer occurs in the presence of a 1,2-d(GpG) cisplatin cross-link.
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DISCUSSION |
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Cisplatin generates a range of bifunctional DNA lesions but our study showed that MutS-specific recognition is limited to the most abundant 1,2-d(GpG) adduct (Table I). The fact that MutS does not recognize specifically a d(ApG) adduct is unexpected because the DNA structure induced by both adducts is considered to be very similar. Indeed these adducts induced a bending and an unwinding of the DNA helix to the same extent (2), but distortions are more pronounced for the 5'-adducted base pair of the 1,2-d(ApG) adduct as compared with the 1,2-d(GpG) cross-link indicating structural differences at the nucleotide level (52). On the other hand, because mispair recognition by MutS was previously shown to be affected by the nature of the flanking base pairs (46, 53), it is possible that the different chemical nature of the 5' base pair of the cross-linked d(GpG) and d(ApG) sites may also play a role. In contrast to the 1,2-d(ApG) adduct, the structures of 1,3-intrastrand and interstrand cross-links differ significantly from that of the major cisplatin lesion (2, 10) and these adducts were not preferentially recognized by MutS (Table I). These data demonstrate the ability of MutS to distinguish between 1,2-d(GpG) intrastrand cross-links and other bifunctional cisplatin lesions and hence illustrate its potential role as a cisplatin DNA damage sensor.
In our assay conditions enabling high affinity and specificity, competition
binding analysis revealed that the 1,2-d(GpG) intrastrand cross-link is poorly
recognized by MutS with a 1.5-fold specificity as compared with an equivalent
unadducted DNA substrate (Fig.
2B). Despite this relatively weak affinity, this binding
might be sufficient for MMR-dependent processing of the 1,2-d(GpG) cross-link
as a G/A mismatch, which shows that similar binding is known to be repaired by
MMR in vitro and in vivo
(46,
53). On the other hand, this
adduct can also interact with a variety of cellular proteins as demonstrated
in eukaryotes (for reviews see Refs.
54 and
55), and as a consequence MMR
binding activity to this adduct might be less favored. In this case, SPR
kinetic data can be helpful to predict the result of the competition between
different cisplatin DNA damage-binding proteins. Indeed, the competition of
two proteins for a platinated DNA substrate has recently been correlated to
their rates of association. Box B of the high mobility group box protein 1
(HMGB1) whose kon is close to the diffusion limit
(109 M-1 s-1)
(56) selectively binds to a
1,2-d(GpG) intrastrand cross-link in the presence of human replication protein
A (57), a DNA damage
recognition protein in the nucleotide excision repair pathway that associates
with this lesion at a rate that is about 2 orders of magnitude lower
(58). In the present study our
SPR assay demonstrated a kon of 3.1 x 104
M-1 s-1 for MutS binding to the 1,2-d(GpG) intrastrand
cross-link in agreement with values published for MutS and eukaryotic
MutS
interacting with various DNA substrates
(50,
59,
60). Thus the kinetic data
obtained with MutS reveal a significantly slower rate of association as
compared with box B of HMGB1 and replication protein A, and so suggest that
these proteins occupy the major cisplatin intrastrand cross-link before the
MMR system can trigger downstream events. In support for this view, the
nucleotide excision repair activity of human cellular extracts with a DNA
substrate containing the major 1,2-intrastrand cross-link was shown to be
unaffected by the presence of hMutS
(13) and a number of cellular
proteins including HMGB, replication protein A, TATA-binding protein, human
upstream binding factor, and Ku autoantigen have been identified in human cell
extracts as cisplatin DNA damage-binding proteins but not mismatch binding
proteins (35,
55).
Several observations both in vitro and in vivo are
consistent with the existence of a variety of cisplatin compound lesions that
are formed during replicative bypass of 1,2-intrastrand cross-links with
misincorporation of a base opposite one of the adducted purines. In the course
of this work, all three possible mispaired bases opposite the adducted purines
(T, G, A and G, A, C opposite the adducted guanines and adenine, respectively)
were tested for binding with MutS. We found that all the compound lesions with
1,2-d(GpG) cross-link and four of six with the 1,2-d(ApG) intrastrand adduct
were specifically recognized by MutS (Table
II). Duplexes containing the 1,2-d(GpG) intrastrand cross-link
were recognized more efficiently when a mispaired base was opposite either
platinated guanine; the ability of the protein to bind DNA substrates with the
1,2-d(ApG) intrastrand adduct was dependent on a mismatched base opposite the
adduct except in two cases. These results are similar to other reports that
have shown that a substrate with O(6)-methylguanine opposite a
thymine is preferentially bound by MutS
(61) and hMutS
(62); ultraviolet light
photoproducts when opposite mismatched bases are also preferentially bound by
hMutS
(43). However, an
unexpected effect reported here is that the 1,2-d(GpG) intrastrand cross-link
results in an increase of MutS mismatch binding activity. Such an effect
occurs when all possible mispaired bases are placed opposite either platinated
guanine except when a mispaired thymine is opposite the 5'-platinated
guanine (Fig. 4,
Table II). Compound lesions
with a 1,2-d(ApG) adduct are less well recognized and the stimulation of
binding they cause is quantitatively less important than that observed with
the 1,2-d(GpG) cross-link. On the other hand, it is noteworthy that the nature
of the mispaired base is also an important parameter for binding to compound
lesions. Indeed, with respect to MutS binding efficiency, compound lesions
follow the same order as the corresponding unplatinated mismatches. These
results are consistent with a mutual influence of both the cisplatin adduct
and the mispair on the recognition of cisplatin compound lesions by MutS.
Taken together, our data clearly show that a large spectrum of cisplatin
compound lesions with both 1,2-intrastrand cross-links are good substrates for
MutS recognition including those that are principally formed in vivo,
namely, compound lesions with an adenine opposite the 5' purine of
either 1,2-intrastrand cross-link. We have yet to determine whether a
cisplatin lesion could activate MutS to engage MMR activity or other
downstream events following the recognition step of a compound lesion.
However, it is conceivable that the significant binding activity of MutS for a
majority of compound lesions and even more for the preference shown for these
compound lesions over mismatched bases could have a bearing on MMR activity,
especially as MutS binding affinity does correspond to the efficiency of
mismatch correction (46).
We cannot exclude the in vivo recognition of compound lesions by
binding proteins other than MutS. In particular, a subset of compound lesions
could be substrates for other mismatch processing pathways. To address this
question, we showed that significant cisplatin compound lesions containing G/A
mispairs are not recognized by the MutY-dependent mismatch repair pathway
indicating that recognition of such lesions could be restricted to MutS.
Similarly, in eukaryotes, binding activity of human cell extracts with two
cisplatin compound lesions (a mismatched thymine opposite either adducted
guanine in a 1,2-d(GpG) adduct) seemed strictly dependent on hMutS
(35). However, repair of these
lesions in human cell extracts seems to argue that proteins of the nucleotide
excision repair pathway may interact with compound lesions
(63). Further studies will be
needed to determine whether proteins from nucleotide excision repair and MMR
compete for binding to cisplatin compound lesions.
Models have been proposed to explain the connection between the MMR pathway
and cytotoxicity of cisplatin
(26,
27,
28,
29). Some of them include
molecular mechanisms initially proposed for recognition of cisplatin DNA
damage by protein such as HMG box proteins. In these models, a first
obligatory step is a relatively tight binding of a protein to a cisplatin
lesion but not to compound lesions. As a consequence, the resulting
nucleoprotein complex could block replication past an adduct leading to either
a cytotoxic response and/or inhibition of the nucleotide excision repair by
shielding the lesion, therefore allowing its persistence in the genome. As
shown in the present study, the fact that MutS recognized poorly the major
cisplatin 1,2-d(GpG) intrastrand cross-link reduces considerably the
likelihood of such models. Our interaction study is more consistent with other
models whereby compound lesions are the critical lesions. A well known model
already proposed for cisplatin and other DNA damaging agents is called the
futile repair model (29). It
was proposed that MMR could be capable of repeatedly initiating repair of the
mispaired base opposite a cisplatin adduct within the newly replicated strand
leading to the accumulation of secondary cytotoxic lesions (DNA strand breaks)
(24,
26). Another attractive model
involves an antirecombinogenic activity of MMR. Such a possibility has been
recently proposed in Saccharomyces cerevisiae and is thought to
involve a RAD52-dependent recombinational bypass during replication
(27). In bacterial cells,
recombination has recently been implicated in the replicative bypass of
cisplatin lesions (64). One
possibility is that MutS binds to cisplatin compound lesions that could be
formed during recombination bypass, leading to inhibition of survival mediated
by recombination-dependent bypass. In this context, the fact that MutS binds
less efficiently to the 1,2-d(ApG) intrastrand adduct than to the 1,2-d(GpG)
intrastrand cross-link when they are part of a compound lesion could
contribute to a higher proportion of error-prone bypass of 1,2-d(ApG) adducts
that could explain in part the higher mutagenic effect of this adduct in MMR
proficient bacterial cells
(14,
15). It would be of interest
to investigate cisplatin mutagenesis in mismatch repair deficient cells to
ascertain such a relationship. As an alternative to the models presented
above, a more direct signaling pathway has been proposed
(28); in such a model, the
assembly of the eukaryotic hMutS and hMutL
proteins at a
specific lesion would then directly trigger apoptotic processes by yet unknown
biochemical events that depend on the presence of a lesion
(62). It would be of interest
to test such a model with various cisplatin compound lesions.
The results presented here have identified critical primary DNA lesions of cisplatin, which recognition is a prerequisite step in the chain of events leading to cisplatin cytotoxicity mediated by MMR. Correlation have been established between loss of MMR in cancer cells and resistance or tolerance to cisplatin. Continued investigations into the molecular mechanisms by which MMR pathways can modulate the cellular response to cisplatin may be helpful to improve the therapeutic use of this drug and in the design of new platinum-based drugs.
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FOOTNOTES |
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Supported by a grant from the Ministère de l'Enseignement
Supérieur et de la Recherche.
¶ On leave at Genoscope from the Centre National de la Recherche
Scientifique. Present address: IntegraGen, 4, rue Pierre Fontaine, 91000 Evry,
France.
|| To whom correspondence should be addressed. Tel.: 33-2-38-25-55-44; Fax: 33-2-38-63-15-17; E-mail: malinge{at}cnrs-orleans.fr.
1 The abbreviations used are: cisplatin,
cis-diamminedichloroplatinum(II); MMR, DNA mismatch repair; RU,
response unit; SPR, surface plasmon resonance; HMG, high mobility group.
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ACKNOWLEDGMENTS |
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REFERENCES |
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