To identify factors that might influence MMR, we used long homonucleotide runs in the yeast Saccharomyces cerevisiae
as reporters of altered MMR capacity. Long homonucleotide runs and other microsatellites are at-risk motifs in MMR-deficient cells6
. These motifs are sensitive reporters for low levels of MMR7-10
and for MMR-deficient cancers3,4,11
because the correction of frequently occurring spontaneous frameshift errors in long homonucleotide runs and microsatellites is accomplished primarily by MMR. Frameshifts in shorter runs and base substitutions are corrected by proofreading as well as by MMR8,12
We chose to test divalent cations as potential inhibitors of MMR because they can affect many enzymatic reactions. Several divalent cations that are also carcinogens can decrease the fidelity of DNA synthesis in vitro13
. Thus, it seemed possible that these cations could also target mutation-avoidance systems in vivo
. We found that chronic exposure to low, non-lethal doses of cadmium (CdCl2
) caused a substantial increase in mutability (as much as 2,000-fold) of long homonucleotide runs in the yeast gene LYS2
( and Supplementary Table 1
online). The strong mutagenic effect was specific to cadmium in that mutagenesis was not detected with other divalent cations or with agents that cause oxidative damage at comparable survival levels (Supplementary Figs. 1
, Supplementary Methods
and Supplementary Table 2
online). In contrast with its strong mutagenic effect, cadmium caused only a small increase in interchromosomal recombination (about 3-fold to 4-fold) and no increase in intrachromosomal recombination (Supplementary Table 3
online), which suggests that mutagenesis does not occur through DNA damage.
Figure 1 The impact of CdCl2 on mutation rates and viability in yeast. Yeast strains are described in Supplementary Methods online. Rates, frequencies and 95% confidence intervals are provided in Supplementary Table 1 online. Frameshift mutation reporters with (more ...)
In addition to causing nuclear mutations, chronic exposure to non-lethal concentrations of cadmium (3 μM and 5 μM) also induced petite mutants (mutants that are unable to grow on a non-fermentable carbon source owing to loss of mitochondrial function; see Supplementary Tables 1
online). Although alteration of mitochondrial function by metal ions could result in reactive oxygen species, mitochondrial loss and even nuclear damage in yeast (ref. 14
and refs. therein), we found comparable levels of cadmium mutagenesis in homonucleotide run reporters in both the wild-type and petite strains (generated by cadmium or by the DNA-intercalating agent ethidium bromide; data not shown). Non-specific changes in gene expression are also an unlikely source of the high mutation frequencies. Although cadmium can alter expression of many proteins in yeast (ref. 15
and refs. therein), deletion of genes associated with cadmium stress, such as MET4, YAP1
, did not alter cadmium mutagenesis in any of the reporters in this study (data not shown).
Because the stability of long homonucleotide runs is extremely sensitive to reduction in MMR capacity, we proposed that MMR itself is a target of cadmium. Mutations that completely eliminate DNA-damage repair pathways have never been reported to cause such strong mutator effects as are caused by MMR defects in long homonucleotide runs and other microsatellites3
. Our experiments with isogenic strains that lack base-excision repair (rad27-Δ16
), nucleotide excision repair (rad1-Δ
, data not shown) and double-strand break repair (rad52-Δ
, data not shown) identified only moderate mutator effects as compared with those caused by defects in MMR. The hypothesis about MMR inhibition was supported by a comparison of the effects of cadmium on frameshift mutagenesis in homonucleotide runs of various sizes and nucleotide content. Cadmium was a strong mutagen for all runs, leading to mutation rates as much as 2,000 times higher (). At the highest concentrations, cadmium-induced mutation rates were 20-50% of those observed in parallel experiments with msh2Δ
strains, which completely lack MMR. There was a notable similarity between cadmium-induced mutation rates and mutation rates in MMR-null strains. In all cases, the cadmium-induced rates (within a group of isogenic strains) were higher for the homonucleotide runs that showed higher mutation rates in the MMR-null background ().
Comparison of cadmium-induced mutation rates in MMR-proficient yeast versus spontaneous mutation rates in MMR-null mutants
If cadmium inhibits MMR such that 20-50% of spontaneous mismatches are left unrepaired, then the high mutation rates characteristic of the MMR-null mutants would not be increased by exposure to cadmium. Consistent with this, the mutation rates in the msh2Δ
mutant for A14 (lys2-A14
) and A7 (his7-2
) homonucleotide runs and the rate of forward mutations in the gene CAN1
were not changed by exposure to cadmium (). We also measured mutation rates in msh6Δ
mutants that are deficient in the MutSα and MutSβ mismatch recognition complexes, respectively. These complexes have partially overlapping function, so that either mutant has a modest mutator phenotype7
. Exposure to 3 μM and 5 μM cadmium increased mutation rates in msh6Δ
mutants to the same levels found in treated wild-type cells (). This suggests that cadmium inhibits both Msh2/Msh6- and Msh2/Msh3-dependent MMR. Using a semi-quantitative test, we also examined cadmium mutagenesis in strains pms1Δ
, which are completely defective in the second step of mismatch recognition3
. The results for mlh1Δ
were comparable to those observed for the msh2
deletion (data not shown).
DNA polymerases Pol δ and Pol ε, which are involved in replicating chromosomal DNA, have intrinsic 3′-to-5′ proofreading exonuclease activity17
. Since a defect in proofreading of either polymerase combined with a MMR defect results in an extremely high mutation rate2,9,18
, we examined the effects of cadmium on proofreading-deficient Pol δ (pol3-01
) and Pol ε (pol2-4
) strains. Cadmium was lethal to pol3-01
haploids but not to pol2-4
(a much weaker mutator) or wild-type haploids (). Isogenic pol3-01
diploids, however, were resistant to cadmium. The lethality caused by cadmium in the Pol δ proofreading-deficient mutant is similar to the lethal effects observed when MMR-null mutations pms1
were introduced into pol3-01
haploids. Double-mutant haploids were inviable owing to the catastrophic accumulation of errors, whereas double-mutant diploids were viable because lethal mutations are generally recessive2,9
Further support for cadmium inhibition of MMR came from examining mutation rates in proofreading-deficient strains (). As expected for a factor that inhibits MMR, cadmium caused a strong increase of mutation rates in proofreading-deficient mutants. This is similar to the synergistic (that is, greater than additive) mutator effects caused by combination of mutated proofreading and MMR alleles. The spontaneous mutation rates in double mutants9,18
were comparable to the cadmium-induced mutation rates in single proofreading-deficient mutants (). As expected with synergy, cadmium-induced mutation rates in the pol3-01
mutants were 2-50 times greater than the sum of mutation rates induced by cadmium in the wild-type strain plus the spontaneous mutation rate in the corresponding proofreading mutant.
The MMR system efficiently repairs not only misalignments that lead to frameshift mutations but also base-base mismatches, thus preventing base substitutions3
. We determined the rates of spontaneous and cadmium-induced frameshifts and base substitutions at the yeast gene CAN1
in the wild type and in a strain deficient in Pol ε proofreading (pol2-4
; ). (Note that the pol2-4
strain has a proofreading-proficient Pol δ.) As expected for MMR inhibition, the rate of frameshift mutation in both strains increased more than the rate of base substitutions, as frameshifts in longer runs are inefficiently prevented by proofreading8,12
. Notably, exposure to cadmium strongly increased not only the rate of frameshift mutation but also the rate of base substitutions, indicating that repair of all kinds of mismatches was inhibited.
Induction of frameshift and base substitution mutations in CAN1 by cadmium
Based on the strong similarity between cadmium mutagenesis and the mutator effects of MMR-null alleles, we conclude that cadmium is a new kind of mutagen that acts by inhibiting the MMR system rather than through DNA damage.
To directly assess the impact of cadmium on human MMR activity, we examined MMR in a human cell extract that can efficiently repair a heteroduplex containing a one-base loop (). Cadmium inhibited the MMR activity in a concentration-dependent manner, with a decrease detectable at 5 μM CdCl2
= 0.05 by Mann-Whitney test). The average decrease of 28% in the in vitro
repair efficiency caused by exposure to 5 μM CdCl2
was similar to the 20-50% of mismatches that were not repaired at comparable concentrations in yeast. The small fraction of unrepaired mismatches (about 2%) that can be detected with the sensitive mutation reporter lys2-A14
in yeast at a concentration of 1 μM CdCl2
was undetectable by the in vitro
assay, because even in the absence of cadmium, MMR in the extract was incomplete. The nature of the in vitro
indicates that the loss of MMR activity must be due to inhibition of one or more steps preceding or including excision. The inhibition was specific to MMR, as cadmium did not interfere with SV40 origin-dependent DNA replication in extracts (data not shown). We did not detect inhibition of MMR with high (50 μM) concentrations of two other divalent cations, cobalt and manganese (data not shown). Further studies are needed to establish if ions other than cadmium can inhibit in vitro
or in vivo
Figure 2 Inhibition of in vitro human strand-specific DNA MMR in a repair-proficient cell extract by cadmium. Data are the average of three independent experiments. Error bars represent standard deviation. The average percent reduction in repair efficiency, taking (more ...)
We suggest that cadmium targets proteins that are directly or indirectly involved in MMR in yeast cells and in human cell extracts. Alteration of protein function has been suggested to explain the toxic, mutagenic and carcinogenic effects of cadmium20-22
. For example, cadmium might bind to cysteine-containing motifs in proteins, such as zinc fingers. Cadmium is also known to bind to calcium channels and calcium-containing proteins. We are currently investigating the possible MMR targets.
The strong mutagenic action of cadmium was observed at concentrations comparable to those found in the environment and at levels that can accumulate in the human body5
. For example, the prostate of healthy unexposed humans accumulates cadmium to concentrations of 12-28 μM and human lungs accumulate cadmium to concentrations of 0.9-6 μM (higher in tobacco smokers23
). Cadmium has only been confirmed as a lung carcinogen but it may also cause prostate cancer5,20
. The primary type of cancer caused by genetic defects in MMR is colorectal cancer4
. There have been some reports identifying a low level of microsatellite instability associated with cancers of the lung11,24
. Further studies are needed to identify conditions and cell types in which cadmium could inhibit human MMR in vivo
(see Supplementary Note
online). Because the level of MMR is an important risk factor in various cancers, even moderate inhibition of MMR has implications for human health. Variations in levels of MMR, possibly due to polymorphisms or differences in MMR levels between tissues or individual cells, could influence the impact of cadmium.
As reduction in MMR has been proposed to facilitate accumulation of adaptive mutations26
, the presence of cadmium in the environment could alter the evolution rate of species with a cadmium-sensitive MMR system. Our findings with cadmium suggest that additional environmental factors could cause genome instability by modulating key DNA metabolic system(s) rather than by damaging DNA. Based on the approaches used in the current study, sensitized systems that include a combination of at-risk motifs and the ability to detect synergistic interactions will provide opportunities to identify other factors that affect the integrity of DNA metabolism.