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.

Impact of the Cellular Zinc Status on PARP-1 Activity and Genomic Stability in HeLa S3 Cells.

Poly(ADP-ribose) polymerase 1 (PARP-1) is actively involved in several DNA repair pathways, especially in the detection of DNA lesions and DNA damage signaling. However, the mechanisms of PARP-1 activation are not fully understood. PARP-1 contains three zinc finger structures, among which the first zinc finger has a remarkably low affinity toward zinc ions. Within the present study, we investigated the impact of the cellular zinc status on PARP-1 activity and on genomic stability in HeLa S3 cells. Significant impairment of H2O2-induced poly(ADP-ribosyl)ation and an increase in DNA strand breaks were detected in the case of zinc depletion by the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) which reduced the total and labile zinc concentrations. On the contrary, preincubation of cells with ZnCl2 led to an overload of total as well as labile zinc and resulted in an increased poly(ADP-ribosyl)ation response upon H2O2 treatment. Furthermore, the impact of the cellular zinc status on gene expression profiles was investigated via high-throughput RT-qPCR, analyzing 95 genes related to metal homeostasis, DNA damage and oxidative stress response, cell cycle regulation and proliferation. Genes encoding metallothioneins responded most sensitively on conditions of mild zinc depletion or moderate zinc overload. Zinc depletion induced by higher concentrations of TPEN led to a significant induction of genes encoding DNA repair factors and cell cycle arrest, indicating the induction of DNA damage and genomic instability. Zinc overload provoked an up-regulation of the oxidative stress response. Altogether, the results highlight the potential role of zinc signaling for PARP-1 activation and the maintenance of genomic stability.