Predicting Novel Arsenic Targets in DNA Repair Pathways

项目摘要 有令人信服的证据表明，抑制DNA修复有助于致癌和共致癌 砷的作用。据报道，两种DNA修复蛋白（PARP-1和XPA）是砷的直接靶点 是因为干扰了锌指功能到目前为止，DNA修复中砷靶点的鉴定 途径仅基于经验证据，尚不清楚是否有其他直接途径。 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3或4个半胱氨酸残基的锌指肽和砷依赖性锌从特异性DNA中的释放 从暴露细胞中分离的C3 H1（例如PARP-1）和C4（例如XPA）修复蛋白，但不包括C2 H2锌指 proteins.这些发现为锌指蛋白的靶向选择性提供了证据，其基于锌指蛋白的数目。 半胱氨酸残基。该项目的目标是实施一个迭代的生物信息学/实验 一种鉴定、测试和改进DNA修复途径中高亲和力砷靶点选择的方法， 为深入了解砷的协同致癌性和DNA修复抑制的机制。这项工作将 产生关于已确定目标的相对敏感性、数量和 半胱氨酸残基在控制观察到的灵敏度中的构型，以及不同锌指的作用 二级结构（如无名指、高音谱号、锌带）在确定砷侵蚀脆弱性方面的作用。 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， 锌指结构和活性与PARP-1或XPA不同，表明砷在体内可能的新作用。 DNA修复抑制。根据我们发表的初步研究结果，我们假设， 生物信息学/实验方法可以被开发和应用于预测高亲和力的砷靶点， 基于锌指构型的DNA修复。为了验证这一假设，我们将：1）确定推定的砷 使用无偏锌指基序模式识别算法的DNA修复靶点，与 来自自动在线数据库检索的结构生物信息学数据和文献注释，以及进一步 通过系统发育和途径分析进行分类。DNA修复途径用作验证集 因为它代表了对砷癌具有直接意义的明确的和生物学相关的焦点 生物学和流行病学。2)使用生物化学和细胞生物学测试亚砷酸盐与预测目标的相互作用 验证潜在目标的方法，确定对砷的相对敏感性，并提供有关 生物信息学方法的迭代改进的结构特征。结果从 拟议的研究预计将通过以下方式推进该领域：1）扩大我们对锌的范围的理解 指状DNA修复蛋白被砷破坏，2）鉴定新的和敏感的靶点，3）建立 特定的锌指结构是否代表优先靶。这些结果将告知可测试的 关于癌症和其他砷相关疾病中其他潜在砷靶点的假设。

Project Summary 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 importance of 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.