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in the Recognition of Iododeoxyuridine in DNA1
Department of Radiation Oncology, Case Western Reserve University, School of Medicine and University Hospitals of Cleveland/Ireland Cancer Center, Cleveland, Ohio 44106
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ABSTRACT |
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(MSH2/MSH6) complex binds specifically to DNA containing an IdUrd-G mismatch, using both purified human MutS as well as nuclear extracts from Msh2-proficient and-deficient mouse cell lines. MutS binding to a IdUrd-G is better recognized than a G-T mismatch in the same sequence context. In addition, MSH2 protein can be found colocalized with IdUrd-DNA using confocal microscopy in G1 synchronized cells after treatment with IdUrd. Consistent with our recent publication, coadministration of IdUrd and a chemical inhibitor of BER, methoxyamine (MX), also increases the extent of MSH2 nuclear colocalization with IdUrd. Furthermore, we show that the extent of MSH2 colocalization with IdUrd in G1-synchronized human tumor cells varies with MLH1 status, suggesting a role for the MLH1 protein in stabilizing the interaction between the MSH2 protein and DNA containing IdUrd. These data, both in vitro and in vivo, suggest direct involvement of MSH2 in processing IdUrd in DNA. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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complex, to bind smaller IDLs, single base-pair mismatches (1
, 3)
, cisplatin adducts (4
, 5)
, O6-methylguanine and O4-methylthymine-containing base pairs (4)
, aminofluorene adducts (6)
, and can also recognize some larger IDLs as well, albeit with much lower efficiency (7)
.
The repair specificities of MutS and MutSß correspond to their binding affinities (2
, 7
, 8)
. The repair efficiency of MutS for single base-pair mismatches is much greater than that of MutSß (9)
, whereas purified MutSß has greater specificity for correcting IDLs ranging from 3 to 10 extra bases (7)
. In addition, the binding affinity and repair efficiency of MutS or MutS directly correlate among various single base-pair mismatch substrates. The MSH2/MSH6 complex has the highest affinity to bind a G-T mismatch, and also repairs this mismatch most efficiently (7, 8, 9)
, with decreasing binding affinity and repair efficiency for six different single base-pair mismatches (9)
.
Localization of the MMR proteins within the cell also corresponds to in vitro binding data and repair assays. In vivo studies have recently shown that both the MSH3 and MSH6 dimeric partners of MSH2 can be localized to DNA replication foci (10)
, consistent with a role for both MutS and MutSß in the repair of replicational errors, and suggesting that it is possible to visualize MMR proteins that localize to areas requiring MMR activity. Additionally, the components of the MutS complex were found to relocate in the cell after treatment with MNNG (11)
, which is known to be selectively cytotoxic to MMR-proficient cells and results in O6-methylguanine-T mispairs in DNA that are recognized by MutS in EMSA studies (4)
.
Previously, we have shown indirect evidence implicating both the MLH1 and MSH2 proteins in processing the halogenated dThd analogues, IdUrd, and BrdUrd in DNA, using multiple genetically matched human and murine cell lines that differ in MLH1 or MSH2 status (12
, 13)
. Our initial studies demonstrated lower levels of IdUrd and BrdUrd in the DNA of MMR-proficient cells, consistent with removal of the drug from DNA by MMR, similar to repair of single base-pair mismatches. However, no significant cytotoxicity nor cell cycle effects were detected after IdUrd treatment, unlike the response to other nucleoside analogues such as 6-thioguanine (12
, 13)
. We now show that human and murine MutS directly bind DNA containing an IdUrd-G mispair with high affinity. We also show nuclear colocalization of MSH2 and IdUrd-DNA in G1-synchronized cells after prior treatment with IdUrd. These data suggest that the nuclear colocalization of MSH2 with IdUrd represents a specific interaction rather than the coincidental presence of the MMR proteins in replication foci with IdUrd. Because recent data from our group also indicate that BER impacts IdUrd-DNA incorporation (14)
, we also studied the effects of a BER inhibitor, MX, on the colocalization of MSH2 with IdUrd. The addition of MX increases the extent of MSH2 colocalization with IdUrd in both MLH1-proficient and MLH1-deficient cell lines. Finally, we also demonstrate that the degree of MSH2 colocalization with IdUrd in G1-synchronized cells correlates with MLH1 status, implicating the MLH1 protein in the stabilization of MSH2 on DNA.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oligonucleotides.
Thirty-one-bp complementary oligonucleotides were obtained from Oligos Etc. Inc. (Wilsonville, OR). They contained a sequence interrupted by either a G-T mismatch or an IdUrd residue paired with various natural bases or a sequence with a G-C complementary base pair, as indicated in Table 1
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and MutSß were generously provided by Dr. Paul Modrich (Duke University Medical Center, Durham, NC). MutS was diluted to a concentration of 50 ng/µl in a buffer containing 25 mM HEPES-KOH (pH 7.5), 330 mM KCl, 1 mM EDTA, 1 mg/ml BSA, and 1 mM DTT. MutSß was diluted to 50 ng/µl in a buffer containing 25 mM HEPES-KOH (pH 7.5), 100 mM KCl, 0.1 mM EDTA, and 2 mM DTT. Nuclear extracts from murine E1A-transformed ES cells from Msh2-/- knockout mice or their Msh2+/+ wild-type siblings were prepared as described previously (13)
. Reactions containing either purified MutS, purified MutSß, or nuclear extract from Msh2+/+ and Msh2-/- cells were preincubated with 1 µg of poly(deoxyiodine-deoxycytidine) on ice for 5 min in a reaction buffer containing 10 mM HEPES (pH 7.6), 5 mM MgCl2, 100 mM KCl, 1 mM DTT, 50 mg/ml BSA, 1 mM EDTA, and 0.4% glycerol. For cold competition experiments with purified MutS, 10–300 ng of unlabeled dsDNA were added during the 5-min preincubation period, and for cold competition with cell extracts, 150 ng of unlabeled dsDNA of the same sequence were added for 5 min at room temperature before the addition of labeled DNA. For supershift of the band visible specifically after incubation with Msh2+/+ nuclear extract, 1 ng of MSH6 (GTBP) monoclonal antibody (Transduction Laboratories, Lexington, KY) was preincubated with nuclear extract from Msh2+/+ cells for 5 min at 37°C, followed by the addition of the labeled probe. Approximately 20 ng of 32P-substrate was then added to the reaction, for a total volume of 20 µl. Samples were then incubated on ice for 10 min (MutS and cell extracts) or 30 min (MutSß), followed by the addition of a loading buffer containing 0.25% bromphenol blue, 0.25% xylene cyanole FF, and 30% glycerol to stop the reaction, and were subsequently loaded onto a 6% nondenaturing polyacrylamide gel at 4°C. Samples were then subjected to electrophoresis at 10 V/cm at 4°C, and dried on a gel dryer at 80° for 2 h. Dried polyacrylamide gels were then exposed to a Molecular Dynamics PhosphorImager Cassette (Molecular Dynamics, Inc., Sunnyvale, CA) at room temperature overnight. Bands were quantitated using Image Quant software (Molecular Dynamics, Inc.).
Nuclear and Cytoplasmic Extract Preparations for Msh2+/+ and Msh2-/- Cells.
Approximately 4 x 106 exponentially growing cells were harvested and centrifuged, and subsequently washed in 1 ml of PBS, then were centrifuged again. The pellet was then washed in a buffer containing 10 mM HEPES (pH 7.4), 10 mM KCl, and 0.5 mM DTT, followed by centrifugation. Cells were then lysed at 4°C in 20 µl of a buffer containing 10 mM HEPES (pH 7.4), 10 mM KCl, 0.5 mM DTT, and 1.0% NP40 for 10 min on ice, with rocking. The nuclei were then collected by centrifugation at 2,000 x g for 5 min, and the supernatant was considered the cytoplasmic extract. The nuclei were then lysed at 4°C for 15 min in a high-salt buffer containing: 20 mM HEPES (pH 7.9), 500 mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 1.5 mM MgCl2, and 20% glycerol, with the use of a Dounce homogenizer as needed, checking for lysis with trypan blue staining under a light microscope. The lysate was then spun down at 12,000 x g for 10 min at 4°C, then was aliquoted and was stored at -80°C.
Fluorescent Staining for IdUrd and MMR Proteins in Synchronized Cells.
Cells were grown to confluence in the presence of 10% FBS or dialyzed FBS (-dThd) with either 10 µM IdUrd alone or 10 µM IdUrd plus 6 mM MX. Cell synchronization in G1 phase was accomplished by incubation with DMEM, supplemented with either 0.1% FBS or 0.1% dialyzed FBS, with treatment as above for 48 h. Cells were released from synchronization by plating onto coverslips in a 6-well dish with DMEM supplemented with 10% FBS. After 5 h, the coverslips were washed with PBS, fixed in 4% formalin solution (Sigma, St. Louis, MO) for 10 min at room temperature, washed three times with PBS, permeabilized by 0.25% Triton X (in PBS) for 5 min at room temperature, washed once with PBS, and then stored at 4°C in PBS before staining. Incubations for blocking and treating with antibodies were done at room temperature for 1 h in PBS/0.3% Tween 20. Coverslips containing fixed cells were blocked in 1% BSA, incubated with a 1:150 dilution of IdUrd monoclonal antibody (CALTAG Laboratories, Burlingame, CA), incubated in antimouse secondary antibody Alexa 568 (Molecular Probes, Eugene, OR) at a dilution of 1:250, incubated with a 1:50 dilution of MSH2 monoclonal antibody (Ab-2; Oncogene Research, Cambridge, MA), and incubated in antimouse secondary antibody Alexa 488 (Molecular Probes) at a dilution of 1:250; wells were washed in PBS/0.3% Tween 20 between each incubation. The coverslips were then mounted on slides with gel-mounting medium containing a nucleus-specific stain (Vector Laboratories, Burlingame, CA) and were placed at -20°C until viewing. The confocal images were obtained at x67 with a Zeiss LSM510 inverted confocal microscope system (Zeiss, Oberkochen, Germany) equipped with a tunable T-Sapphire laser (Mira-F-V5-XW-220) with a diode pump laser (Verdi 5 W) to obtain the images with the different dyes at the Confocal Core Microscopy Facility at Case Western Reserve University. Image analysis was carried out with Adobe Photoshop software (Adobe Systems, San Jose, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recognizes the dThd Analogue IdUrd in DNA. to bind to dsDNA containing the physiologically relevant base pair IdUrd-A and IdUrd-G (18
, 19)
, as well as IdUrd-C and IdUrd-T (Fig. 1)
. A 31-bp dsDNA probe containing a G-T mismatch in the middle, shown previously to be bound with high affinity by MutS (20)
, was used as a positive control for binding. The same sequence was also used to compare MutS binding to a G-C complementary base-pair or a base pair containing IdUrd, at the same position as the G-T mismatch (Table 1)
. In Fig. 1a
, Lane 2, incubation of MutS with heteroduplex DNA results in an intense band, representing the specific shift of the 32P-probe, compared with the very low level of background binding to a complementary base pair within the same sequence (Fig. 1a
, Lane 8). Similar to results with Taq MutS (17)
, human MutS does not bind dsDNA containing IdUrd paired with adenine with greater affinity than to homoduplex DNA in the same sequence context (Fig. 1a
, Lane 4). However, when IdUrd is mispaired with a G, the MutS dimer shows a very strong shift (Fig. 1a
, Lane 6), indicating high-affinity binding. A single-stranded oligonucleotide containing IdUrd (oligonucleotide c, Table 1
) was also incubated with purified MutS to determine whether IdUrd alone was recognized by the MMR complex, but MutS did not show any binding to this substrate (data not shown). Binding of MutS was also tested for a 31-mer containing IdUrd-C and IdUrd-T base pair (Table 1)
, which are thought to occur infrequently when IdUrd is incorporated into DNA (19
, 21)
. MutS recognizes both of these substrates to a degree comparable with homoduplex DNA in the same sequence context (Fig. 1b)
, suggesting that the substrate that the MutS complex recognizes after incorporation of IdUrd into DNA is an IdUrd-G mismatch. This correlates with the high affinity of this complex both for naturally arising G-T mismatches (3)
and for other chemically modified bases and drug-induced DNA adducts, including O6-methylguanine-T pairs in DNA (4)
, S6-methylthioguanine-T pairs in DNA (22)
, cisplatin adducts (4
, 5)
, and aminofluorene adducts (6)
.
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and MutSß, as mutation spectrums indicate (23)
, and repair of some single-base mismatches by MutSß can occur in in vitro repair assays (9)
. As a result, we also used EMSA analysis with IdUrd in the same sequence context used for the MutS binding, using dsDNA containing a (CA)5 IDL (Table 1)
as a positive control for binding and found that MutSß does not recognize IdUrd-G pairs in DNA (data not shown).
An IdUrd-G mismatch is also selectively recognized by proteins in nuclear extracts of murine MMR-proficient cells (Fig. 2)
. We found that there are two retarded bands after incubation of a double-stranded fragment of DNA containing an IdUrd-G mismatch with Msh2+/+ nuclear extract that are not present after incubation with Msh2-/- nuclear extract (Fig. 2
, Lanes 2 and 3). In addition, one of these bands is clearly immunoreactive with the antibody to the MSH6 protein (Lane 4), suggesting that MSH6 is present in a complex with dsDNA containing an IdUrd-G pair, causing the 32P-probe to migrate more slowly. Cold competition with excess unlabeled dsDNA of the same sequence context, containing an IdUrd-G base pair, or a G-T mismatch (Table 1)
, also abolishes the retarded bands specific to the Msh2+/+ nuclear extract (Lanes 5 and 6), with cold IdUrd-G more effectively abolishing the two bands than cold G-T. This also corresponds to Fig. 3
, which demonstrates that MutS has a higher affinity for an IdUrd-G pair in this sequence context than for a G-T pair in this sequence. Excess unlabeled dsDNA of the same sequence, but containing a G-C base pair rather than an IdUrd-G pair, does not eliminate the two retarded bands specific to the Msh2+/+ nuclear extract (Fig. 2
, Lane 7), further supporting the possibility that the two retarded bands specific to the Msh2+/+ nuclear extract are the result of MMR proteins binding to the dsDNA containing an IdUrd-G base pair. Also, the addition of all three unlabeled dsDNA fragments results in the abolishment of bands common to both Msh2-/- and Msh2+/+ extracts, demonstrating that these retarded bands are not the result of specific recognition by the MMR proteins (Lanes 5–7).
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Complex for a G-T Mismatch and an IdUrd-G Mismatch in DNA. for an IdUrd-G mispair with that of the recognition complex for a G-T mismatch, we used various amounts of purified MutS with equivalent amounts of each 31-mer. In three separate reactions, the MutS complex consistently demonstrated higher specific binding activity for an oligonucleotide containing an IdUrd-G mismatch than for a G-T mismatch (Fig. 3, a and b)
. This can also be demonstrated using unlabeled oligonucleotides containing a complementary G-C base pair, a G-T mismatch, or an IdUrd-G mismatch to compete for binding by MutS to a 32P-labeled 31-mer containing an IdUrd-G pair. Although preincubation with unlabeled G-C does not affect MutS binding to an IdUrd-G mismatch (Fig. 3c)
, preincubation with both unlabeled and heteroduplex oligonucleotides quickly eliminates the binding to IdUrd-G. However, cold IdUrd-G competes for binding at lower concentrations than does cold G-T; and at the highest concentrations, preincubation with cold IdUrd-G prevents all binding to the labeled IdUrd-G oligonucleotide, whereas preincubation with the same concentration of cold G-T does not. It is likely that the alteration in DNA, and possibly the charge distribution created by an IdUrd-G mispair, is slightly different from that caused by the presence of a G-T mismatch, affecting the binding specificity of MutS for these different substrates.
Nuclear Colocalization of MSH2 with IdUrd.
The endogenous MSH2 protein is distributed throughout the nucleus and cytoplasm of intact HCT116/3-6 and HCT116 human colon cancer cells in the absence of treatment (Fig. 4, b and k
, respectively). These data are in contrast with previous publications in which these proteins were found to be exclusively nuclear in intact cells among multiple tissue types (24
, 25)
but are consistent with in vitro MMR assays using cytoplasmic extract (10)
, as well as with a more recent report that demonstrates the presence of MSH2 and MSH6 in the cytoplasm of HeLa cells using both Western blot analysis and immunocytochemical staining of intact cells (11)
. Because a short pulse (15 min) with the halogenated dThd analogue BrdUrd has been used to colocalize MSH3 and MSH6 to replication foci, we have looked for MSH2 colocalization with IdUrd in a synchronized population of cells in G1. We found that MSH2 remains colocalized with IdUrd during G1 (Fig. 4)
, in which cell cycle status was confirmed with flow cytometry (data not shown), indicating that the colocalization cannot be attributed exclusively to the presence of MSH2 in replication foci. We also found that MSH2 colocalized with IdUrd in both MLH1-deficient and MLH1-proficient cells, although the extent of colocalization is greater in MLH1-proficient HCT116/3-6 cells (compare Fig. 4o
with Fig. 4f
). These data demonstrate that MLH1 is not necessary for the nuclear colocalization of MSH2 and IdUrd but suggest that MLH1 may be necessary for a prolonged interaction with, or for a stable MMR complex assembly on, DNA that contains IdUrd.
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, suggesting that there are greater numbers of IdUrd residues available for binding by MSH2 in the presence of MX, which would otherwise be bound by BER proteins. These data on combined IdUrd + MX treatment are consistent with our recently published study showing higher levels of IdUrd-DNA incorporated (as measured by HPLC) compared with IdUrd alone (14)
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DISCUSSION |
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and nuclear colocalization of the MSH2 protein with IdUrd, supporting a direct role for the MSH2 protein in the recognition of IdUrd in DNA. In agreement with previous studies of the Taq MutS protein (17)
, we find that human MutS does not recognize DNA that contains IdUrd-A pairs but does recognize IdUrd-G mispairs in the same sequence context with high affinity, indicating that this is the primary substrate for the MMR-associated differences in IdUrd levels in DNA (12
, 13)
. Previous studies using chemically modified bases and single base-pair mismatches in the same location, within the same sequence context that we have used in our EMSA analysis, have demonstrated that the human MutS complex recognizes a G-T mismatch with greater specificity than do aminofluorene and 2-acetylaminofluorene adducts (6)
, cisplatin adducts (5)
, and O6-methylguanine- or O4-methylthymine-containing pairs (4)
at the same position. Therefore, an IdUrd-G mismatch appears to be unique in that it is recognized by the human MutS complex in this same sequence context more efficiently than a G-T mismatch. Of the possible base pairings between Watson-Crick bases and IdUrd, an IdUrd-G pair would bear the greatest similarity to a G-T mismatch in DNA. However, the van-der-Waals radius of iodine is just slightly larger than that of the methyl group at the same position of dThd (26)
, which may cause a greater distortion of the DNA helix containing IdUrd-G than would a G-T mismatch and could explain the higher affinity of MutS for DNA that contains IdUrd-G than for DNA that contains G-T in the same sequence context. Previous studies with the structurally similar dThd analogue, BrdUrd, suggest that base pairing of this analogue with guanine does occur in vivo in 
phage and Escherichia coli, and plays a role in BrdUrd-induced mutagenesis (19
, 21)
.
We have also shown that MutS has very little affinity for IdUrd paired with adenine, cytosine, or thymine in the same sequence context, suggesting that MMR is not involved in processing these pairs in DNA. This would also correlate with our previous data, in which we have shown that, although the levels of IdUrd in DNA decrease over time in MMR-proficient cells, approximately 5.0% of the dThd remains substituted with IdUrd in most cell lines that we have studied (12
, 13)
. It is likely that IdUrd pairs with other bases in addition to guanine, part of the time, and we speculate that these pairs probably account for the analogue that remains in the DNA of MMR-proficient cells.
Our binding data indicate that MMR proteins can recognize DNA that contains IdUrd-G pairs with high affinity and specificity, and this is supported by protein colocalization of MSH2 and IdUrd in nuclear foci of synchronized cells in G1. Our data from the synchronous G1 cells indicates that the nuclear localization of MSH2 is not the result of the incidental presence of the protein in replication foci. This is interesting also because these G1 foci may be indicative of active MMR of DNA that contains IdUrd, because it has been demonstrated that strand-specific DNA MMR can be carried out in cells at various stages of the cell cycle, such as in G2-synchronized cells (27) , in G1 synchronized cells (28) , and in extracts of senescent, mitotic, and postmitotic tissues from Drosophila (29) .
It is also important to consider the potential role of BER in processing IdUrd in DNA, based on our recent publication (14)
. In the present study, we demonstrate that treatment with the BER inhibitor MX increases the extent of MSH2 colocalization with IdUrd (Fig. 4)
. Although MX is known to inhibit single-nucleotide BER by blocking repair of the AP site after glycosylase-mediated removal of a base (30)
, this MX/AP stable intermediate is known to be a substrate for long-patch BER (31)
. Our results using confocal microscopy in this study and using HPLC analysis in the prior study (14)
show that there are a greater number of IdUrd residues in DNA after cotreatment with MX, which suggests that, although IdUrd does not appear to be a substrate for removal by single-nucleotide BER, it is likely a substrate for long-patch BER. Consistent with this, addition of MX during treatment with IdUrd results in an increase in the extent of MSH2 colocalization with IdUrd. This would indicate that long-patch BER also plays a role in binding IdUrd in DNA and thereby prevents MSH2 recognition of some IdUrd residues.
In conclusion, although BER also is likely to play a role in the recognition and processing of IdUrd in DNA, these data demonstrate that MMR proteins directly interact with DNA that contains IdUrd, and that IdUrd-G mispairs are likely to be the primary substrate for recognition by MutS. Our findings also demonstrate that the interaction of the MMR proteins with IdUrd is prolonged and can occur during G1, and that MLH1 is not necessary for the specific interaction of MSH2 and IdUrd but may stabilize it. These data provide more direct evidence for the interaction of MMR proteins with IdUrd and shed light on one mechanism for the cellular processing of IdUrd after incorporation into DNA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA50595 and CA84578 (to T. J. K.). 
2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Lerner Tower 6068, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-6068. Phone: (216) 844-2530; Fax: (216) 844-4799; E-mail: timothy.kinsella{at}uhhs.com
3 The abbreviations used are: MMR, mismatch repair; dThd, thymidine; IdUrd, iododeoxyuridine; BrdUrd, bromodeoxyuridine; BER, base excision repair; IDL, insertion-deletion loop; MNNG, N-methyl-N'-nitro-N–nitrosoguanidine; EMSA, electrophoretic mobility shift assay; MX, methoxyamine; HPLC, high-performance liquid chromatography; dsDNA, double-stranded DNA.
Received 2/18/03. Revised 4/24/03. Accepted 6/11/03.
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REFERENCES |
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recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct. Proc. Natl. Acad. Sci. USA, 93: 6443-6447, 1996. specifically binds to DNA containing aminofluorene and acetylaminofluorene adducts. J. Biol. Chem., 271: 24084-24088, 1996.. J. Biol. Chem., 273: 19895-19901, 1998. phage: 5-bromouracil and hydroxylamine. Mol. Gen. Genet., 152: 29-36, 1977.[Medline]
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