Predicting Novel Arsenic Targets in DNA Repair Pathways

描述（由申请人提供）：有令人信服的证据表明，DNA修复的抑制有助于砷的致癌和共致癌作用。两个DNA修复蛋白（PARP-1和XPA）已被报道为砷干扰锌指功能的直接靶点。迄今为止，鉴定DNA修复途径中的砷靶点完全基于经验证据，尚不清楚是否存在其他直接和高度敏感的DNA修复靶点。我们最近的工作证明了亚砷酸盐与含有3或4个半胱氨酸残基的锌指肽的优先相互作用，以及从暴露于C3H1（如PARP-1）和C4（如XPA）细胞中分离的特定DNA修复蛋白中释放的亚砷酸盐依赖性锌，而不是C2H2，锌指蛋白。这些发现为锌指蛋白基于半胱氨酸残基数量的靶向选择性提供了证据。本项目旨在通过生物信息学/实验方法对DNA修复通路中高亲和力砷靶点进行鉴定、测试和优化选择，以深入了解砷共致癌性和DNA修复抑制机制。这项工作将产生关于已确定目标的相对敏感性的关键信息，半胱氨酸残基的数量和结构在控制观察到的敏感性方面的重要性，以及不同锌指二级结构（如环指，高音谱号，锌带）在确定砷攻击脆弱性方面的作用。利用生物信息学方法的初步结果发现了含有锌指结构和活性不同于PARP-1或XPA的新的候选DNA修复靶点，提示砷可能在DNA修复抑制中的新作用。基于我们已发表的和初步的研究结果，我们假设可以开发一种生物信息学/实验相结合的方法，并应用于基于锌指结构的DNA修复中高亲和力砷靶点的预测。为了验证这一假设，我们将：

DESCRIPTION (provided by applicant): There is compelling evidence that inhibition of DNA repair contributes to the carcinogenic and co-carcinogenic actions of arsenic. Two DNA repair proteins (PARP-1 and XPA) have been reported as direct arsenic targets based on interference with zinc finger function. To date, identification of arsenic targets in DNA repair pathways has been based solely on empirical evidence and it is unknown whether there are additional direct and highly sensitive DNA repair targets. Our recent work demonstrates preferential interaction of arsenite with zinc finger peptides containing 3 or 4 cysteine residues and arsenite-dependent zinc release from specific DNA repair proteins isolated from exposed cells in C3H1 (e.g. PARP-1) and C4 (e.g. XPA), but not C2H2, zinc finger proteins. These findings provide evidence for target selectivity of zinc finger proteins based on the number of cysteine residues. The objective of this project is to implement an iterative bioinformatic/ experimental approach to identify, test and refine the selection of high-affinity arsenic targets in the DNA repair pathway, in order to gain insights into mechanisms of arsenic co-carcinogenicity and DNA repair inhibition. This work will yield critical information on the relative sensitivities of identified targets, the importanceof number and configuration of cysteine residues in governing observed sensitivities, and the role of distinct zinc finger secondary structures (e.g. ring finger, treble clef, zinc ribbon) in determining vulnerability to arsenic attack. Preliminary results using a bioinformatic approach identified novel candidate DNA repair targets containing zinc finger structures and activities distinct from PARP-1 or XPA, suggesting possible new actions of arsenic in DNA repair inhibition. Based on our published and preliminary findings, we hypothesize that a coupled bioinformatic/ experimental approach can be developed and applied to predict high affinity arsenic targets in DNA repair, based on zinc finger configuration. To test this hypothesis we will: 1) Identify putative arsenic targets in DNA repair using an unbiased zinc finger motif pattern recognition algorithm, correlated with structural bioinformatic data and literature annotations from automated online database searches, and further classified through phylogenetic and pathway analyses. The DNA repair pathway is used as the validation set since it represents a well-established and biologically-relevant focus of direct significance to arsenic cancer biology and epidemiology. 2) Test arsenite interaction with predicted targets using biochemical and cell biology approaches to validate potential targets, establish relative sensitivities to arsenic, and provide information on structural characteristics for iterative refinement of the bioinformatics approach. The outcomes from the proposed studies are expected to advance the field by 1) expanding our understanding of the scope of zinc finger DNA repair protein disruption by arsenic, 2) identifying novel and sensitive targets, and 3) establishing whether specific zinc finger structures represent preferential targets. These results will inform testable hypotheses regarding additional potential arsenic targets in cancer and other arsenic-associated diseases. PUBLIC HEALTH RELEVANCE: Given the widespread public exposure to arsenic in municipal and private water supplies, there is interest and concern in observations that arsenic concentrations at or near the EPA maximum contaminant level greatly enhance the carcinogenic potential of other DNA damaging agents and inhibit DNA repair. Thus, arsenic may contribute to elevated cancer risk when individuals are exposed to other carcinogens through occupational, environmental or lifestyle exposures. This project represents the first effort to computationally predict highly sensitive arsenic targets to better understand the impact of arsenic on DNA repair and inform strategies to reverse or prevent the adverse health effects of arsenic exposure in humans.