Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex

Abstract

Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2-MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2-MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2-MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2-MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.

Figure 1

Effect of cadmium on the ATPase activity of MSH2–MSH6. (a) Effect of 100 μM Cd²⁺ on increasing concentrations (0–160 nM) of MSH2–MSH6. Pre-incubation of Cd²⁺ with MSH2–MSH6 (black bars) at 4°C for 10 min followed by incubation with the reaction mixture containing ATP at 37°C for 30 min. Direct incubation of MSH2–MSH6 (gray bars) with Cd²⁺ and ATP in reaction mixture at 37°C for 30 min without any pre-incubation. ATPase activity of MSH2–MSH6 in the absence of Cd²⁺ (white bars). Each bar corresponds to the average of three experiments. Standard deviation is indicated at the top of each bar. (b) Effect of increasing concentrations of Cd²⁺ (0–0.5 mM) on 160 nM (closed squares) and 80 nM (closed triangles) of MSH2–MSH6. Pre-incubation of MSH2–MSH6 with Cd²⁺ at 4°C for 10 min was carried out. The average of three experiments for each concentration of Cd²⁺ is presented for MSH2–MSH6 at 160 nM and the average of four experiments for MSH2–MSH6 at 80 nM. Standard deviation is included. The curves were not fitted. (c) Time-course study over 120 min pre-incubation of the ATPase activity of MSH2–MSH6 (160 nM) in the presence (closed squares) and the absence of Cd²⁺ (open triangles). The average of three experiments for each time point is presented. Standard deviation is included. Curves were fitted by linear regression analysis.

Figure 2

Effect of cadmium on the DNA-binding activity of MSH2–MSH6. (a) MSH2–MSH6 was pre-incubated with Cd²⁺ at 4°C for 10 min, mixed with reaction mixture containing labeled G/T mispaired DNA and further incubated at 4°C for 15 min in 20 μl final volume. Gel was run with 10 μl of the mixture, and autoradiographed. Quantitation was carried out with a phosphorImager. (b) Comparison of the ATPase activity (white bars) and DNA mobility shift activity (black bars) of MSH2–MSH6 (80 nM) in the presence of 0–0.5 mM Cd²⁺. Activity is presented as a percentage of the activity of MSH2–MSH6 in the absence of Cd²⁺ taken as 100%. The average of three experiments for each concentration of Cd²⁺ is presented. Bars also include standard deviation.

Figure 3

Effect of cadmium on the ATP hydrolysis and ATP-binding activities of MSH2–MSH6. MSH2–MSH6 was pre-incubated with Cd²⁺ (0–0.5 mM) at 4°C for 10 min. Aliquots were withdrawn and assayed for ATP hydrolysis (black bars) and ATP binding (white bars) as described in Materials and Methods. Results are expressed as a percentage of the activity of MSH2–MSH6 in the absence of Cd²⁺. For ATP hydrolysis 100% corresponds to 200 pmol ATP hydrolyzed, while for ATP binding, 100% of the activity corresponds to 2 pmol ATP bound. The average of three experiments for each concentration of Cd²⁺ is presented. Standard deviation is included.

Figure 4

Effect of zinc on the cadmium-induced inhibition of MMR. (a) Yeast mutator assay using a strain carrying the lys2-10A allele was preformed in the presence of 1 μM Cd²⁺ and 0–1000 μM Zn²⁺. The appearance of Lys⁺ revertant colonies indicates a mutator phenotype. (b) Effect of Zn²⁺ on the mutator rate of the lys2-10A strain in the presence of Cd²⁺. Each bar corresponds to the average of three sets of experiments using five independent colonies per set. Rates are calculated as described in Materials and Methods and standard deviation is included at the top of each bar. (c) MSH2–MSH6 (40 nM) was pre-incubated with 50 μM Cd²⁺ (2/6+Cd), 50 μM Zn²⁺ (2/6+Zn) or a combination of both (2/6+Cd+Zn) at 4°C for 10 min and assayed for ATPase activity as described in Materials and Methods. To remove excess metal ions, the mixture was dialyzed (denoted by a D in the Figure) extensively and assayed for ATPase activity. (2/6+Cd)D indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis; (2/6+Zn)D indicates that MSH2–MSH6 was treated with Zn²⁺ followed by dialysis; (2/6+Cd+Zn)D indicates that MSH2–MSH6 was treated with Cd²⁺ and Zn²⁺ followed by dialysis; and (2/6+Cd)D+Zn indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis, and then treated with Zn²⁺. A comparison of the ATPase activity of the dialyzed and undialyzed MSH2–MSH6 is shown. The activity of untreated, undialyzed MSH2–MSH6 was used as 100% and corresponds to 26 pmol ATP hydrolyzed. Each set of experiments was repeated three times and the average is presented together with the standard deviation.

Figure 5

Role of sulfhydryl groups as targets in the inhibition of the ATPase activity of MSH2–MSH6 by cadmium. (a) Comparison of the inhibition of ATPase activity by NEM and Cd²⁺. MSH2–MSH6 (40 nM) was pre-incubated with 0.5 mM Cd²⁺ (2/6+Cd), 0.5 mM NEM (2/6+NEM) or a combination of both (2/6+Cd+NEM) at 4°C for 10 min and assayed for ATPase activity. To remove excess NEM and Cd²⁺, the pre-incubation mixture was extensively dialyzed (denoted as D in the Figure) and assayed for ATPase activity.(2/6+Cd)D indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis; (2/6+NEM)D indicates that MSH2–MSH6 was treated with NEM followed by dialysis; and (2/6+Cd)D+NEM indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis, and the treated with NEM. The comparison of the ATPase activities is shown here. The average of three experiments is presented, bars include the standard deviation. (b) Effect of DTT (2 mM) on the inhibition of the MSH2–MSH6 ATPase activity by Cd²⁺ (0.5 mM) and NEM (5 mM). MSH2–MSH6 (160 nM) was pre-incubated with Cd²⁺ for 15 min (2/6+Cd), Cd²⁺ for 15 min followed by DTT for 15 min(2/6+Cd+DTT), DTT for 15 min followed by Cd²⁺ for 15 min (2/6+DTT+Cd), NEM for 15 min (2/6+NEM), NEM for 15 min followed by DTT for 15 min (2/6+NEM+DTT), and DTT for 15 min followed by NEM for 15 min(2/6+DTT+NEM). The mixtures were then assayed for ATPase activity as described in Materials and Methods. The average of three experiments is presented, including the standard deviation.