Metal inhibition of the base excision repair enzymes

描述（由申请人提供）：已知金属会导致人类癌症，一般人群不可避免地接触到各种金属，如Cd2+， Ni2+和Pb2+。然而，金属致癌的分子机制尚不清楚。最近的数据表明，DNA修复的抑制可能是致癌和突变的一个重要因素。本研究的长期目标是通过抑制DNA修复来确定金属毒性的分子机制。金属毒性代表了一种新的机制，通过这种机制，各种环境诱变剂和致癌物可以破坏DNA的完整性，从而导致细胞中的诱变和致癌事件。该领域的研究主要集中在利用金属依赖的催化机制或需要金属离子来维持结构完整性的修复酶的子集上。本应用程序的目的是在碱基切除修复（BER）酶中确定潜在的抑制靶点，这些酶的功能和/或结构不需要金属。中心假设是金属离子，如Cd2+， Ni2+和Pb2+可以通过结合关键活性位点残基或改变催化反应的化学性质来干扰需要修复的非金属酶的催化活性。暴露于这些金属会抑制酶对其有毒/诱变DNA底物的活性。抑制关键的DNA损伤修复途径实际上可能比致癌物和/或诱变剂直接损伤DNA更重要。我们最近获得的数据表明，BER酶之一，人类n -甲基嘌呤- dna糖基酶（MPG）被几种有毒金属离子抑制，这一假设是基于这些数据制定的。我们还观察到无细胞提取物对尿嘧啶- dna糖基酶（UNG）活性的抑制，这表明另一种非金属需要酶是金属抑制的目标。这一假设将通过两个特定目标进行验证：1)使用计算方法（QM/MM）揭示金属离子与BER修复酶相互作用的机制；2)使用生化分析确定金属离子抑制BER糖基酶的新靶点，这些酶的结构或功能不需要金属离子。这项研究具有重要意义，因为它将导致鉴定DNA修复蛋白中金属离子抑制的新靶点。它还将导致这些金属和修复蛋白之间分子相互作用的新的结构和机制细节。最终，通过这项努力揭示的机制将有助于未来的研究开发潜在的预防和/或治疗方法，以阻止或消除这些有毒金属与生物学重要分子的相互作用。此外，拟议的研究将提高私家侦探的竞争力和生产力，使他能够发展成为一名独立的研究者，并实现以下发展目标：(a)建立一个独立的研究小组，(b)提高指导技能，以及(c)提高研究质量和专业发展。

DESCRIPTION (provided by applicant): Metals are known to cause cancer in humans and the general population is unavoidably exposed to various metals, such as Cd2+, Ni2+ and Pb2+. However the molecular mechanisms of metal carcinogenesis are still unknown. Recent data suggest that inhibition of DNA repair may be an important contributing factor to carcinogenesis and mutagenesis. The long-term goal of this research is to identify molecular mechanisms of metal toxicity through the inhibition of DNA repair. Metal toxicity represents a novel mechanism, by which various environmental mutagens and carcinogens can destabilize DNA integrity leading to mutagenic and carcinogenic events in the cell. Research in this area has focused on the subset of repair enzymes that utilize a metal-dependent catalytic mechanism or require a metal ions for maintaining structural integrity. The goal of this application is to identify potential inhibition targets among the base excision repair (BER) enzymes which do not require metals for their function and/or structure. The central hypothesis is that metal ions, such as Cd2+, Ni2+ and Pb2+ can interfere with the catalytic activity of non-metal requiring repair enzymes through binding to key active site residues or changing the chemical nature of the catalytic reaction. Exposure to these metals leads to the inhibition of enzyme activity towards its toxic/mutagenic DNA substrates. Inhibition of key DNA damage repair pathways may actually be even more important than direct DNA damage by carcinogens and/or mutagens. The hypothesis has been formulated based on our recently obtained data showing that one of the BER enzymes, human N-methylpurine-DNA glycosylase (MPG), is inhibited by several toxic metal ions. We also observed inhibition of the Uracil-DNA glycosylase (UNG) activity in cell-free extracts suggesting that another non-metal requiring enzyme is a target for metal inhibition. This hypothesis will be tested by two specific aims: 1) Use computational approaches (QM/MM) to uncover the mechanisms of metal ion interactions with BER repair enzymes, 2) Identify new targets for metal ion inhibition among BER glycosylases that do not require metal ions for their structure or function using biochemical assays. The proposed research is significant, because it will lead to the identification of new targets for metal ion inhibition among DNA repair proteins. It will also lead to new structural and mechanistic details on molecular interactions among these metals and the repair proteins. Ultimately, the mechanisms uncovered through this effort will aid future studies to develop potential prevention and/or treatment approaches to block or remove these toxic metal interactions with biologically important molecules. In addition the proposed research will increase the competiveness and productivity of the PI allowing him to develop into an independent investigator and achieve the following developmental objectives: (a) establish an independent research group, (b) enhance mentoring skills, and (c) improve the quality of research and professional advancement. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will examine the toxic effects of metal ions on reducing the capacity of DNA repair, which will lead to an increase in carcinogenic and mutagenic events in the cell. The general population is exposed to toxic metals through occupational exposure, food, water, air and a variety of consumer products. Understanding the mechanisms, by which toxic metals interfere with DNA repair, is relevant to developing fundamental knowledge that will aid in cancer prevention and treatment.