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。两种DNA修复蛋白(PARP-1和XPA)已被报道为直接的砷靶标 基于对锌指功能的干扰。到目前为止，识别DNA修复中的砷靶标 途径仅基于经验证据，目前尚不清楚是否存在其他直接的 和高度敏感的DNA修复目标。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多肽与特定DNA亚砷酸盐依赖的锌释放 从C3H1(如PARP-1)和C4(如XPA)暴露的细胞中分离出修复蛋白，但不包括C2H2、锌指 蛋白质。这些发现为基于锌指蛋白数量的靶向选择性提供了证据 半胱氨酸残留物。该项目的目标是实施迭代的生物信息学/实验 在DNA修复途径中识别、测试和改进高亲和力砷靶标选择的方法 目的：了解砷协同致癌和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、数量和重要性的关键信息 半胱氨酸残基在控制观察到的敏感性中的构型，以及不同的锌指的作用 二级结构(如无名指、高音谱带、锌带)在确定砷攻击的易感性方面的作用。 利用生物信息学方法识别新的候选DNA修复靶点的初步结果 锌指结构和活性不同于PARP-1或XPA，提示砷在 DNA修复抑制。根据我们已发表的和初步的发现，我们假设一个耦合 生物信息学/实验方法可用于预测高亲和力砷靶标 DNA修复，基于锌指构型。为了验证这一假设，我们将：1)确定推定的砷 在DNA修复中使用无偏锌指基序模式识别算法的目标，相关 来自自动化在线数据库搜索的结构化生物信息数据和文献注释，以及进一步 通过系统发育和通径分析进行分类。将DNA修复路径用作验证集 因为它代表了一个对砷癌具有直接意义的公认的、与生物相关的焦点 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，并提供关于 生物信息学方法迭代求精的结构特征。结果来自于 拟议的研究有望通过1)扩大我们对锌的范围的了解来推动该领域的发展 砷对手指DNA修复蛋白的破坏，2)寻找新的敏感靶点，3)建立 特定的锌指结构是否代表优先靶标。这些结果将通知Testable 关于癌症和其他砷相关疾病的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.