Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.

Recent studies in yeast have found that processing of DNA double-strand breaks (DSB) for recombination repair involves Sgs1 helicase. Human cells have five Sgs1 homologues with unknown selectivity and significance for repair of different DSB types. Here we examined the importance of WRN helicase in repair of G(2)-specific DSB caused by abnormal mismatch repair (MMR) of ternary Cr-DNA adducts. We found that Cr(VI) induced a rapid dispersal of WRN from the nucleolus resulting in its prolonged retention in the nucleoplasm. The loss of MSH2 or MLH1 MMR proteins abolished the long-term but not the initial WRN relocalization. WRN-deficient fibroblasts were hypersensitive to Cr(VI)-induced clonogenic death and contained high levels of persistent DSB detected by gamma-H2AX/53BP1 foci and pulsed-field gel electrophoresis. WRN was involved in recombination repair of Cr-induced DNA damage, as evidenced by WRN-RAD51 colocalization and defective formation of RAD51 foci in the absence of WRN. The accumulation of unrepaired DSB in WRN-depleted cells was rescued by the inactivation of MMR, indicating that MMR-generated DSB were a key substrate for WRN action in Cr(VI)-treated cells. Competition for the limited amounts of WRN in primary cells between G(2) processes of telomere rebuilding and recombinational repair is expected to increase persistence of Cr-induced DSB and may cause telomeric abnormalities in tissues of chronically chromate-exposed workers. Our work provides the first demonstration of the major importance of WRN in repair of a specific class of DSB in human cells.

ABSTRACT Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (Tn OtChr ) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long Tn OtChr of the mixed Tn 21 /Tn 3 transposon subfamily contains a group of chrB , chrA , chrC , and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC , were essential for establishment of high resistance in chromium-sensitive O. tritici . The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA . Expression of chrB , chrC , or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. Tn OtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to Tn OtChr -containing cells in the environments with multiple redox-active contaminants.

The biomolecular mechanism of nickel carcinogenicity is driven by intracellular Ni cation. The role of nickel catalyst in single-wall carbon nanotube toxicity will therefore depend on its bioavailability, which is highly uncertain due to encapsulation by carbon shells. This article measures the material-specific Ni release into extra- and intracellular physiological fluid phases and suggests practical techniques for managing the metals contribution to SWNT toxicity.