Metal inhibition of the base excision repair enzymes

描述(申请人提供)：已知金属会致癌，普通人群不可避免地接触到各种金属，如Cd~(2+)、Ni~(2+)和Pb~(2+)。然而，金属致癌的分子机制仍不清楚。最近的数据表明，DNA修复的抑制可能是癌症发生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完整性，导致细胞内发生诱变和致癌事件。这一领域的研究主要集中在修复酶的子集上，这些修复酶利用金属依赖的催化机制或需要金属离子来保持结构完整性。这项应用的目的是在碱基切除修复(BER)酶中识别潜在的抑制靶点，这些酶的功能和/或结构不需要金属。中心假说是，金属离子，如Cd~(2+)、Ni~(2+)和Pb~(2+)可以通过结合关键活性中心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。接触这些金属会导致对其有毒/诱变的DNA底物的酶活性的抑制。抑制关键的DNA损伤修复途径实际上可能比致癌物和/或诱变剂对DNA的直接损伤更重要。这一假说是基于我们最近获得的数据提出的，该数据表明，BER酶之一，人N-甲基嘌呤-DNA糖基酶(MPG)，被几种有毒金属离子抑制。我们还观察到无细胞提取物中Uracil-DNA糖基酶(UNG)的活性受到抑制，这表明另一种非金属要求的酶是金属抑制的目标。这一假设将通过两个特定的目标得到验证：1)使用计算方法(QM/MM)来揭示金属离子与BER修复酶相互作用的机制；2)利用生化分析在不需要金属离子的BER糖基酶中寻找金属离子抑制的新靶点。这项研究具有重要意义，因为它将导致在DNA修复蛋白中识别新的金属离子抑制靶点。它还将导致关于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，通过这一努力发现的机制将有助于未来的研究，以开发潜在的预防和/或治疗方法，以阻断或消除这些有毒金属与生物重要分子的相互作用。此外，拟议的研究将提高私人助理的竞争力和生产力，使他能够发展成为一名独立的调查员，并实现以下发展目标：(A)建立一个独立的研究小组，(B)加强指导技能，以及(C)提高研究的质量和专业晋升。 公共卫生相关性：拟议的研究与公共健康相关，因为它将检查金属离子对降低DNA修复能力的毒性影响，这将导致细胞中致癌和突变事件的增加。普通民众通过职业接触、食物、水、空气和各种消费品接触有毒金属。了解有毒金属干扰DNA修复的机制，对于发展有助于癌症预防和治疗的基础知识是相关的。

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.