Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.

Arsenite Impairs BRCA1-Dependent DNA Double-Strand Break Repair, a Mechanism Potentially Contributing to Genomic Instability.

BRCA1 is a key player in maintaining genomic integrity with multiple functions in DNA damage response (DDR) mechanisms. Due to its thiol-rich zinc-complexing domain, the protein may also be a potential target for redox-active and/or thiol-reactive (semi)metal compounds. The latter includes trivalent inorganic arsenic, which is indirectly genotoxic via induction of oxidative stress and inhibition of DNA repair pathways. In the present study, we investigated the effect of NaAsO2 on the transcriptional and functional DDR. Particular attention was paid to the potential impairment of BRCA1-mediated DDR mechanisms by arsenite by comparing BRCA1-deficient and -proficient cells. At the transcriptional level, arsenite itself activated several DDR mechanisms, including a pronounced oxidative stress and DNA damage response, mostly independent of BRCA1 status. However, at the functional level, a clear BRCA1 dependency was observed in both cell cycle regulation and cell death mechanisms after arsenite exposure. Furthermore, in the absence of arsenite, the lack of functional BRCA1 impaired the largely error-free homologous recombination (HR), leading to a shift towards the error-prone non-homologous end-joining (NHEJ). Arsenic treatment also induced this shift in BRCA1-proficient cells, indicating BRCA1 inactivation. Although BRCA1 bound to DNA DSBs induced via ionizing radiation, its dissociation was impaired, similarly to the downstream proteins RAD51 and RAD54. A shift from HR to NHEJ by arsenite was further supported by corresponding reporter gene assays. Taken together, arsenite appears to negatively affect HR via functional inactivation of BRCA1, possibly by interacting with its RING finger structure, which may compromise genomic stability.