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DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.
Cisplatin (cis-diaminodichloroplatinum (II)) is an antitumor agent which has a cure rate of greater than 90% for testicular cancer . It reacts with DNA to produce mostly intrastrand crosslinks between adjacent guanines (65% of the total), adjacent guanine and adenines (25%), and guanines separated by a base (1,3-GNG, 5–10%) [2, 3]. Interstrand crosslinks comprise about 2% of the total adducts and small amounts of monoadducts are also formed. The biologically inactive trans isomer of cisplatin also produces 1,3-GNG adducts, interstrand crosslinks and monoadducts [4, 5], suggesting that intrastrand crosslinks between adjacent purines are the biologically important adducts of cisplatin since they efficiently block progression of DNA polymerases in vitro and in vivo .
Nucleotide excision repair (NER) removes platinated intrastrand crosslinks but the 1,3-GNG lesions are removed at a rate 50-fold faster than those between adjacent purines . The importance of NER is manifested by the increased sensitivity of NER-deficient mutants of Escherichia coli and mammalian cells to cisplatin [8–10]. In addition, however, recombinational repair mechanisms are as important as NER in allowing cells to survive cisplatin damage since E. coli strains in which recombination is defective show increased susceptibility to cisplatin [11, 12]. Cisplatin is also a potent inducer of recombination .
Methylating agents are used in cancer chemotherapy and laboratory versions, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), react with DNA and cause a signature type of damage, producing a variety of lesions which include O6-methylguanine (O6-meG), 3-methyladenine and 7-methylguanine . These lesions are usually removed either directly, in the first case by a methyltransferase, or through base excision repair using specific glycosylases.
GATC sequences in E. coli DNA are methylated directly after replication at the N6 position of adenine by Dam methyltransferase a product of the dam gene . The DNA behind the replication fork is already methylated on the parental strand but not on the newly-synthesized daughter strand to give hemimethylated DNA. Replication errors are present in the unmethylated strand and MMR is initiated when MutS binds to base mismatches . Recruitment of MutL and MutH forms a ternary complex which activates the latent endonuclease activity of MutH to cleave 5’ to the G at an unmethylated GATC sequence. The UvrD helicase unwinds the unmethylated strand in either the 3’ or 5’ direction and which is digested by exonucleases with a distinct polarity. Resynthesis is accomplished by the polymerase III holoenzyme complex and the resulting nick is ligated by DNA ligase . In wildtype cells, mismatch correction is restricted to the hemimethylated region as MutH has little, if any, activity on fully methylated DNA . In dam mutants, which lack DNA adenine methyltransferase, no methylation is present and MMR can occur in newly replicated as well as in unreplicated DNA because MutH can utilize unmethylated DNA as a substrate .
E. coli dam mutants are more sensitive to the cytotoxic effects of cisplatin  and MNNG [20, 21] than wildtype. Mutations disabling the MMR system (mutS, mutL) in a dam cell, however, render it as resistant to these agents as wildtype [19, 20]. The MutS protein from E. coli specifically recognizes the platinated GG intrastrand crosslink opposite CC or CT [22, 23] and O6-meG-cytosine and O6-meG-thymine base pairs [23, 24] and the latter can be formed by replicative or bypass polymerase action . MMR action on cisplatin intrastrand crosslinks in dam bacteria leads to the formation of double-strand breaks that require recombination to repair them . MutS interferes with RecA-mediated strand exchange during the recombinational repair process .
Although dam bacteria are sensitive to the cytotoxic action of both MNNG and cisplatin via specific binding by MutS, the recognition of adducts by this enzyme is complex. We recently described a mutated MutS protein, MutSΔ800, produced from a multicopy plasmid, that sensitizes dam cells to MNNG but not to cisplatin, and was found to bind to O6-meG mismatches but not platinated GG intrastrand crosslinks opposite CC . MMR in wild type cells occurs near the replication fork and the MutS protein has been reported to interact with the DNA polymerase III beta-clamp  which confers processivity on the holoenzyme by "clamping" the catalytic subunit, DnaE, to DNA. Sensitivity of dam cells to cisplatin and MNNG could, therefore, reflect an interaction between mismatch repair and DNA polymerase III holoenzyme and associated replication proteins. We have examined various DNA replication mutants and we describe two instances where dna gene products alter the cellular response to the two agents. Furthermore, if the interaction between the beta clamp, a product of the dnaN gene, and MutS is diminished it might lead to a mutator phenotype due to ineffective MMR. We show that the dnaN159 mutant does indeed have a mutator phenotype but it is not due to lack of MMR.
The bacterial strains used in this study are listed in Table 1. J. R. Walker's laboratory  constructed and designated strain GM36 (dnaX36), but it is called GM8051 in this paper to avoid confusion with the strain of the same name but different genotype derived in this laboratory. The dnaX plasmid, obtained from J.R. Walker (University of Texas, Austin), is a derivative of pBR322 .
L medium contains 20 g tryptone (Difco), 10 g yeast extract (Difco), 0.5 g NaCl, 4 ml of 1M NaOH per liter and solidified when required with 16 g of agar. Brain Heart Infusion (Difco) broth was prepared using 20 g of powder per liter of water and solidified, when required, with 16 g of agar . Minimal medium was prepared as described by Davis and Mingioli  and supplemented with amino acids (80 µg/ml) as required. Ampicillin and rifampicin were included in media at 100 µg/ml while, tetracycline and chloramphenicol were included in media at 10 µg/ml. Kanamycin was included, when required, at 20 µg/ml. Media for the reversion assays contained 10% lactose and Xgal (40 ng/ml) in minimal media.
Multiple cultures (generally 10) of the deletion strains and their isogenic partners were generated from an inoculum of a few hundred cells and grown to saturation. Portions of the cultures were plated on BH medium with rifampicin and incubated at 30°C and 34°C or 37°C depending on cell viability, until colonies appeared. Media were also supplemented with 100 ug ampicillin /ml when required.
CC101–107 dnaN159 derivatives were constructed by P1vir transduction using the closely-linked zid-501::Tn10 marker. CC101–106 mutS458 derivatives have been described  and CC107 mutS458 was constructed by P1 vir transduction. Ten cultures of these strains and the CC101–107 parental strains were grown to saturation in minimal media. For Fig. 6, 10 ul of the culture was spotted on a minimal media plate containing lactose and Xgal and incubated at 30°C until colonies formed and papillae appeared. For Table 3, portions of the cultures were plated and incubated at 30°C on the same type of plates and blue colonies counted to calculate the reversion rate. Viable counts were determined on glucose minimal plates.
Cisplatin (Sigma) was dissolved in water and incubated at 37°C for at least 2 hours. The molar concentration was measured by taking an absorbance spectrum and reading a maximal absorbance at 301nm and dividing by the extinction coefficient, 131 M−1 cm−1. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG (Sigma)) was prepared by dissolving 1 mg of MNNG in 100 ul of DMSO and adding 900 ul of sterile water. Cells were grown in 10 ml L medium to an OD600 of 0.35–0.45, harvested and resuspended in the same volume of minimal salts. Varying concentrations of cisplatin were added and incubated at 30°C for 1 hour. Serial dilutions of cisplatin-exposed cells were plated on L media plus ampicillin and incubated overnight. For MNNG exposure, the logarithmic phase cells in L broth were exposed to various concentrations of MNNG for 10 minutes at 30°C followed by dilution for plating as described above.
At the permissive temperature for growth, 30°C, the dnaE486 (encodes the alpha-catalytic subunit of PolIII) strain is more sensitive than wildtype to cisplatin, but not MNNG (Fig. 1). The degree of cisplatin sensitivity of the dnaE486 strain is the same as that for a dam-13 mutant (Fig. 1A). The dnaE486 dam-13 double mutant strain is more sensitive to both cisplatin and MNNG than either parent.
The dnaX36 (tau and gamma subunits of PolIII) bacteria show the same survival to cisplatin as wildtype at 30°C but are more sensitive at 37°C to both cisplatin and MNNG (Fig. 2) although the viable counts of these cultures were the same at the two temperatures. An unusual feature of DnaX is that when expressed from a multicopy plasmid at 30°C, the dnaX36 host strain bearing it becomes sensitive to cisplatin (Fig. 2A), to the same degree as a dam mutant, but remains resistant to MNNG (Fig. 2B). No sensitivity to cisplatin was detected when the same plasmid is expressed in a wildtype strain (Fig. 2A) or with the vector plasmid (data not shown). We could not test the effect of the dam-13 dnaX36 combination because although the strain was viable at 30°C, it cannot form colonies at 37°C in contrast to the parental strains.
There was no differential effect on the dnaN159 (beta subunit of PolIII holoenzyme) strain to the cytotoxic action of cisplatin and MNNG (Fig. 3). The dam-13 dnaN159 double mutant, however, is more resistant to MNNG than the dam-13 strain alone (Fig. 3B). This increased resistance could be due to a reduction in MMR since null mutations in the mutS or mutL genes in a dam mutant increase resistance to MNNG to the wildtype level. To monitor the effect of MMR on dnaN159 dam-13 survival, a dnaN159 dam-13 mutS458 triple mutant was constructed. The survival of the double and triple mutants after MNNG exposure was the same (Fig. 3B) suggesting a reduction of MMR in the dnaN159 dam-13 strain (see Discussion).
Neither dnaB43 (helicase) nor dnaG3 (primase) mutants showed a differential sensitivity to cisplatin or MNNG (Figs, 4, ,5).5). The dam derivatives of these strains showed no additional increase in sensitivity to both agents indicating that the dna strains had a wildtype phenotype at the temperature (30°C) at which the experiments were conducted. The exception was the dam dnaG3 strain which showed increased sensitivity to MNNG but not cisplatin (Fig. 4B).
Bacteria with an inactive MutS protein exhibit a mutator phenotype . If the interaction between the MutS protein and the beta-clamp, a product of the dnaN gene , is required for efficient MMR, then if it is diminished a mutator phenotype should result. We therefore tested the parental and dnaN temperature sensitive mutant strain as well as dnaB, dnaE, dnaG and dnaX bacteria for spontaneous mutant frequency to rifampcin-resistance at the permissive temperature, 30°C. The results in Table 2 show that dnaN has the highest mutation frequency, a 35-fold increase, in comparison to the parental strain. The dnaX mutant has a wildtype level of spontaneous mutagenesis at 30°C but this is increased to 10-fold at 37°C, a temperature at which full viability is retained. A strain with a holD mutation, which encodes the psi subunit of the holoenzyme, has been reported to show a seven-fold increase in spontaneous mutation frequency using rifampicin-resistance as an assay . Bacteria with the dnaE486 allele show a 10-fold increase in mutation frequency to rifampicin resistance (Table 2) confirming a previous 40-fold increase with this marker  and a 6-fold increase in his-4 reversion . The dnaB and dnaG mutants do not exhibit a mutator phenotype (Table 2), as there is less than a 2-fold difference when comparing these strains with the wild type strain.
To determine the mutation spectrum in the dnaN159 strain, we have used the lacZ reversion assay described by Cupples and Miller [38, 39]. The dnaN159 mutation was introduced into each of the seven tester strains and the number of Lac+ revertants was measured at 30°C and compared to the wildtype strains. The results are shown in Table 3 and Fig. 6. There is a 7-fold increase in AT to TA transversions which is probably due to increased SOS induction in this strain [36, 40]. This change is not expected in MMR-deficient strains which show increases in transition and +1 and −1 frameshift mutations [32, 38, 39] and the pattern of reversion of a mutS strain is clearly different from that of dnaN159 (Fig. 6). We did not observe an increase in GC to AT revertants in the mutS strain which was expected based on previous results [33, 38, 41]. We conclude that the increased spontaneous mutation frequency in the dnaN159 mutant is not due to diminished MMR.
We showed previously that the mutSΔ800 mutation on a multicopy plasmid in a mutS null mutation host conferred resistance to cisplatin, but not MNNG, and that there was reduced binding of MutSΔ800 to platinated, but not methylated, oligonucleotides . The contrasting types of modifications produced in DNA by these agents prompted us to examine if DNA polymerase III holoenzyme mutants also showed such a differential sensitivity. The results are summarized in Table 4. We found that unlike wildtype, dnaE486 bacteria (Fig. 1) and those overproducing DnaX (Fig. 2) were more sensitive to cisplatin but resistant to MNNG. Possible explanations are that the DnaE486 holoenzyme (at the permissive temperature) is less capable of restarting replication after removal of a blocking cisplatin crosslink than a DnaE holoenzyme during chromosome replication. This holoenzyme might not be as efficient as DnaE in gap filling during MMR. Alternatively, the DnaE486 holoenzyme progression may not be blocked as efficiently as DnaE by cisplatin adducts leading to fork disintegration. On the other hand, an O6-meG base pair, which is a miscoding but not a blocking lesion, would be replicated by both DnaE486 and DnaE holoenzymes with, presumably, similar efficiencies.
The DnaX overproducing plasmid in adnaX36 host restores the ability to grow at 43°C indicating complementation of the mutant phenotype by the wildtype gene product (Fig. 2). Although complementation by DnaX overproduction also restores MNNG resistance to the wildtype level, the strain is still sensitive to cisplatin. There are at least two possible explanations. First, since nothing is known about the regulation of DNA polymerase III holoenzyme assembly, it is possible that overproduction of the tau and gamma subunits, the products of the dnaX gene, disrupts holoenzyme function. This option is unlikely because overproduction of DnaX in the wildtype does not lead to cisplatin sensitivity. Second, it is possible that the DnaX36 clamp loader complex has a higher affinity for other holoenzyme proteins, such as DnaN (beta clamp), and although DnaX is overproduced clamp loader complexes of DnaX and DnaX36 are formed. Since DnaX36 is probably slightly defective, the mixed DnaX-DnaX36 complex is proficient for clamp loading onto methylated but not platinated DNA.
Dam mutS bacteria are resistant to cisplatin and MNNG. MutS interacts with the beta clamp  and this interaction may be important for the re-synthesis step of MMR by DNA polymerase III holoenzyme. If this interaction is critical for MMR sensitization, then an increase in resistance to cisplatin and MNNG might be expected in the dam-13 dnaN159 mutant. At the permissive temperature, there was no difference in survival between the double mutant and the dam-13 strain to cisplatin (Fig. 3). The dam-13 dnaN159 strain, however, is more resistant to the cytotoxic effect of MNNG than dam-13 bacteria which indicates reduced MMR perhaps due to a reduction in MutS-beta clamp interaction. The triple dam-13 dnaN159 mutS458 mutant has the same survival as the dam-13 dnaN159 double mutant suggesting that indeed there is reduced MMR (Fig. 3). The triple mutant is expected to have the same resistance as wild type because that is observed in dam mutS strains. This is not the case indicating that there must also be an MMR-independent sensitivity to MNNG in the dnaN159 strains as well as an MMR impairment.
Although reduced MMR in the dnaN159 strain affects survival after exposure to MNNG, it is not sufficient to affect the mutant frequencies shown in Tables 2 and and33 and Fig. 6. That is, the pattern of reversion is clearly not that expected for an MMR-deficient strain. The dnaN159 mutant displays constitutive SOS induction and this is the likely explanation for the increase in AT to TA transversions . The reduction in +1 frameshift mutations in CC107 dnaN159 and GC to TA transversions in CC104 dnaN159 could be due to slower DNA replication resulting in more efficient repair of replication errors (frameshifts) and spontaneous oxidative lesions (8-oxoguanine) . For the latter, it is also possible that SOS-induced translesion polymerases in the dnaN159 strain efficiently bypass this modified base, while in the wildtype it is mutagenic when replicated by DNA polymerase III holoenzyme.
Mammalian cells are sensitive to the cytotoxic action of MNNG  and cisplatin  but MMR deficient cell lines derived from them are resistant to the action of these agents, although this association for cisplatin has recently been questioned [44, 45]. The MutS protein from human cells specifically recognizes the platinated GG intrastrand crosslink and O6-meG/cytosine and thymine base pairs . The sensitization by MMR to cisplatin or MNNG has been proposed to occur by several models including futile repair cycles by MMR at lesions followed by double-strand break formation and subsequent signaling for cell cycle arrest and apotosis . An alternative model posits a direct link between lesion recogntion by MutS and a signal transduction cascade leading to cell death [47, 48]. Insofar as the work reported here with E. coli can be extrapolated to human cells, our results would favor the double-strand break model.
We thank E.A. Adelberg, C. Cupples, J.R. Walker and J. Wechsler for donating the bacterial strains and plasmids listed in Table 1. We thank Mike Volkert, Anetta Nowosielska and Mark Sutton for suggestions to improve the manuscript. This work was supported by grant GM63790 from the National Institutes of Health.