Predicting Novel Arsenic Targets in DNA Repair Pathways

描述（由申请方提供）：有令人信服的证据表明，抑制DNA修复有助于砷的致癌和共致癌作用。两种DNA修复蛋白（PARP-1和XPA）已被报道为基于干扰锌指功能的直接砷靶点。到目前为止，DNA修复途径中砷靶点的鉴定仅基于经验证据，尚不清楚是否存在其他直接和高度敏感的DNA修复靶点。我们最近的工作表明，优先相互作用的亚砷酸盐与锌指肽含有3或4个半胱氨酸残基和砷依赖性锌释放从特定的DNA修复蛋白分离出暴露的细胞在C3 H1（如PARP-1）和C4（如XPA），但不是C2 H2，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选择性提供了证据。该项目的目标是实施迭代生物信息学/实验方法，以识别，测试和改进DNA修复途径中高亲和力砷靶点的选择，以深入了解砷的致癌性和DNA修复抑制机制。这项工作将产生关键信息的相对灵敏度确定的目标，importanceof的数量和配置的半胱氨酸残基在管理观察到的灵敏度，以及不同的锌指二级结构（如无名指，高音谱号，锌带）在确定砷攻击的脆弱性的作用。使用生物信息学方法的初步结果确定了新的候选DNA修复靶点，其中含有与PARP-1或XPA不同的锌指结构和活性，这表明砷在DNA修复抑制中可能具有新的作用。基于我们已发表的初步研究结果，我们假设可以开发一种耦合的生物信息学/实验方法，并应用于基于锌指构型预测DNA修复中的高亲和力砷靶点。为了验证这个假设，我们将： 1)使用无偏锌指基序模式识别算法识别DNA修复中的推定砷靶点，与自动在线数据库搜索的结构生物信息学数据和文献注释相关，并通过系统发育和途径分析进一步分类。DNA修复途径被用作验证集，因为它代表了对砷癌生物学和流行病学具有直接意义的成熟和生物学相关的焦点。2)使用生物化学和细胞生物学方法测试亚砷酸盐与预测目标的相互作用，以验证潜在目标，建立对砷的相对敏感性，并为生物信息学方法的迭代改进提供结构特征信息。这些研究的结果有望通过以下方式推动该领域的发展：1）扩大我们对锌指DNA修复蛋白被砷破坏的范围的理解，2）确定新的和敏感的靶标，3）确定特定的锌指结构是否代表优先靶标。这些结果将为关于癌症和其他砷相关疾病中其他潜在砷靶点的可验证假设提供信息。 公共卫生相关性：鉴于广泛的公众暴露于砷在市政和私人供水，有兴趣和关注的观察，砷浓度或接近EPA的最大污染物水平，大大提高了其他DNA损伤剂的致癌潜力，并抑制DNA修复。因此，当个体通过职业、环境或生活方式暴露于其他致癌物质时，砷可能会导致癌症风险升高。该项目首次尝试通过计算预测高度敏感的砷目标，以更好地了解砷对DNA修复的影响，并为扭转或预防人类砷暴露对健康的不良影响提供策略。

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.