This project is in response to NOT-OD-09-058 """"""""NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications"""""""" and represents an extension of R01 ES015826 (K.J. Liu, PI;L.G. Hudson, co-investigator) """"""""Arsenic-enhanced skin carcinogenesis by UV radiation"""""""". In vitro and in vivo carcinogenicity studies provide strong support for the conclusion that metals, including arsenite, can act as co-carcinogens when combined with non metal carcinogens. Although inhibition of DNA repair by arsenite is consistent with the hypothesis that arsenic acts as a co-carcinogen, the actual mechanisms are unclear. Proposed protein targets of arsenite include Poly (ADP-ribose) polymerase (PARP)- 1, a protein with two C3H1 zinc finger motifs in the DNA binding domain and one C4 zinc binding motif responsible for DNA-dependent catalytic activity. Using PARP-1 as a model protein based on its exquisite sensitivity to arsenite and its role in DNA strand break/base excision repair pathways, we will probe the mechanism of arsenite-dependent PARP-1 inhibition using an innovative approach combining functional, cellular, and analytical methodologies at the interface of cell biology and chemistry. We will focus on binding of trivalent arsenicals to cysteine residues in the zinc binding motifs and test the mechanistic prediction that vicinal cysteine residues are required for high affinity arsenite binding, thus C3H1 and C4 zinc binding motifs are more likely targets for arsenite interaction. Although arsenic-dependent inhibition of DNA repair enzymes has been reported, this chemical mechanism has received little attention. We hypothesize that arsenite inhibits PARP-1 activity and PARP-1 dependent DNA repair through disruption of zinc finger function via a mechanism involving direct displacement or replacement of zinc by arsenite. We will test this hypothesis by introducing specific Cys/His substitutions in the zinc binding motifs of PARP-1. We will measure the affinity of arsenite and zinc for each of the zinc finger polypeptides using analytical techniques including MALDI-TOF-MS, cobalt spectophotometry and ICP-MS (Aim 1), and further test the functional impact of these modifications on PARP- 1 activity using site-directed mutagenesis (Aim 2). The proposed studies will significantly increase our knowledge regarding arsenic interactions with a prototype zinc finger protein. This chemical biology approach will provide important insight to the molecular mechanisms of how arsenite modifies zinc fingers, thus inhibiting the function of zinc finger containing proteins. The findings from these studies are likely to lead to testable hypotheses regarding additional direct protein targets for arsenite and guide future studies to investigate other key zinc finger proteins relevant to arsenite toxicity.
The NIEHS Strategic Plan specifically notes """"""""the study of environmental factors that modify DNA damage, repair, and maintenance is an important area of investigation, particularly with regard to aging, cancer, and cell death."""""""" Epidemiological evidence supports that environmental or occupational exposures to arsenic contributes to numerous acute and chronic toxic effects in humans including carcinogenesis, and the carcinogenicity of arsenic is believed to be due to arsenic-induced DNA damage and arsenic-dependent inhibition of DNA repair. Gaining a detailed molecular understanding of the mechanisms by which arsenic inhibits DNA repair activities through disruption of zinc finger motifs will be relevant to understanding arsenic interactions with other potential zinc finger protein targets and inform strategies to overcome or ameliorate excess arsenic exposures.
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