STRUCTURAL STUDY OF A DNA ADDUCT DEVIRED FROM A TUMORIGENIC METABOLITE OF BENZO

苯并致瘤代谢产物 DNA 加合物的结构研究

• 批准号: 7956119
• 负责人: Suse Broyde
• 金额: $ 0.08万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956119
• 关键词: Active Sites; Affect; Aromatic Polycyclic Hydrocarbons; Automobile Exhaust; Bacillus (bacterium); Base Sequence; Benzo(a)pyrene; Biomedical Research; Breathing; Bypass; Carcinogens; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Adducts; DNA biosynthesis; DNA-Directed DNA Polymerase; Enzymes; Epoxy Compounds; Family; Fishes; Frequencies; Funding; Genes; Glycols; Grant; Guanine; High Performance Computing; High temperature of physical object; Human; Institution; Lead; Lesion; Meat; Modeling; Molecular; Molecular Conformation; Mutation; Oncogenes; Outcome; Polymerase; Relative (related person); Research; Research Personnel; Resources; Site; Source; Structural Models; Structure; System; Temperature; Tobacco smoke; Tumor Suppressor Genes; United States National Institutes of Health; adduct; base; cancer initiation; conformer; flexibility; meetings; member; research study; tumorigenic

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Current evidence suggests that bulky carcinogen-DNA adducts are bypassed by DNA polymerases through a polymerase switch model [1-5]. In this model the replicative DNA polymerases carry out DNA replication with high fidelity and efficiency until they meet the carcinogen-damaged sites in DNA. However, they are frequently blocked by such lesions. After the replicative DNA polymerase is dissociated from the replication fork, a bypass polymerase may be called in to replicate past the lesion. At a very low frequency, the replicative DNA polymerase itself can also bypass the lesion [5-9], presumably if the adduct is in certain permissive conformations. Lesion bypass by both types of polymerases can be either mutagenic or non-mutagenic. Mutations will occur if a mismatched partner is incorporated opposite the adduct or slippage of the primer strand relative to the template has occurred when the polymerase is attempting to transit the lesion. Such mutations, if occurring in certain critical genes such as oncogenes or tumor suppressor genes, can lead to cancer initiation [10]. Polycyclic aromatic hydrocarbons (PAHs) are environmental pro-carcinogens that are produced during combustion of organic materials. Benzo[a]pyrene (BP) is one of the most extensively studied PAHs, and is usually found in a wide range of substances ingested or inhaled by humans, such as automobile exhaust, tobacco smoke and broiled meats and fish [11-13]. It can be metabolically activated to a number of metabolites including (+)-anti-BPDE (benzo[a]pyrene diol epoxide), which is highly mutagenic and tumorigenic in mammalian systems [14, 15]. The metabolites can attack DNA and the base primarily attacked is guanine; a 10S(+)-trans-anti-[BP]-dG ([BP]G) adduct is predominantly formed [16-19]. Experimental studies [20] have demonstrated that the 10S(+)-trans-anti-[BP]-dG adduct mainly blocks a bacterial replicative DNA polymerase, Bacillus fragment (BF) [21], with very little bypass. However, this same adduct is more easily bypassed by an archaeal bypass DNA polymerase, Dpo4 which is a member of the DinB family also found in humans [22]. In addition, base sequence context (CG*G vs. TG*G, G*=10S(+)-trans-anti-[BP]-dG) has been shown to affect bypass efficiency of Dpo4 [20]. Base sequence context effects on mutagenicity are important in relation to understanding surprisingly different mutagenic outcomes in different sequence contexts. Furthermore, in the case of BF and Dpo4, greater bypass efficiency is observed at 55C, compared to 37C [20]. High temperature is studied due to the fact that both BF and Dpo4 are thermophilic enzymes whose efficiency is greatest at higher temperature. We hypothesize that the observed different replicating activities of BF and Dpo4 for this adduct are due to structural differences between them, especially at the active site, and that high temperature enhances the flexibility of the polymerase, thereby making less favored conformers of the adduct more accessible. In order to investigate the structural factors responsible for the different activities of the two enzymes in replicating the BP modified DNA, we will carry out the following studies in pursuit of two specific aims: Specific aim 1: For BF, create dynamic structural models of the [BP]G adduct in open binary and closed ternary complexes at pre-insertion, insertion and post-insertion sites, to explain observed blockage and rare bypass at low and high temperatures Specific aim 2: For Dpo4, create dynamic structural models of the [BP]G adduct in binary and ternary complexes at the insertion site to explain the easier bypass, the observed sequence context effect, and the temperature effect Our proposed specific aims will provide the molecular details that connect function with structure.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 目前的证据表明，DNA 聚合酶通过聚合酶开关模型绕过了大体积的致癌物-DNA 加合物 [1-5]。在该模型中，复制性 DNA 聚合酶以高保真度和效率进行 DNA 复制，直到遇到 DNA 中致癌物质损伤的位点。然而，它们经常被此类病变阻塞。复制DNA聚合酶从复制叉解离后，可能会调用旁路聚合酶来复制穿过病变。在极低的频率下，复制 DNA 聚合酶本身也可以绕过损伤 [5-9]，大概是在加合物处于某些允许构象的情况下。两种类型的聚合酶绕过病变可以是诱变的或非诱变的。如果在加合物对面掺入了不匹配的配偶体，就会发生突变，或者当聚合酶试图穿过病变时，引物链相对于模板发生滑移。如果此类突变发生在某些关键基因（例如癌基因或肿瘤抑制基因）中，则可能导致癌症发生[10]。多环芳烃 (PAH) 是有机材料燃烧过程中产生的环境致癌物。苯并[a]芘（BP）是研究最广泛的多环芳烃之一，通常存在于人类摄入或吸入的多种物质中，例如汽车尾气、烟草烟雾以及烤肉和鱼[11-13] 。它可以被代谢激活为许多代谢物，包括 (+)-抗 BPDE（苯并[a]芘二醇环氧化物），它在哺乳动物系统中具有高度诱变性和致瘤性 [14, 15]。代谢物可以攻击DNA，主要攻击的碱基是鸟嘌呤；主要形成 10S(+)-反式-抗-[BP]-dG ([BP]G) 加合物 [16-19]。实验研究[20]表明，10S(+)-反式抗[BP]-dG加合物主要阻断细菌复制DNA聚合酶、芽孢杆菌片段(BF)[21]，几乎没有旁路。然而，这种加合物更容易被古菌旁路 DNA 聚合酶 Dpo4 绕过，Dpo4 是 DinB 家族的成员，也在人类中发现 [22]。此外，碱基序列上下文（CG*G 与 TG*G、G*=10S(+)-trans-anti-[BP]-dG）已被证明会影响 Dpo4 的旁路效率 [20]。碱基序列环境对致突变性的影响对于理解不同序列环境中令人惊讶的不同致突变结果非常重要。此外，就 BF 和 Dpo4 而言，与 37C 相比，在 55C 时观察到更高的旁路效率 [20]。研究高温是因为 BF 和 Dpo4 都是嗜热酶，其在较高温度下效率最高。我们假设观察到的 BF 和 Dpo4 对于该加合物的不同复制活性是由于它们之间的结构差异，特别是在活性位点，并且高温增强了聚合酶的灵活性，从而使加合物的不太受欢迎的构象异构体更容易接近。为了研究两种酶在复制 BP 修饰 DNA 时具有不同活性的结构因素，我们将开展以下研究，以实现两个具体目标： 具体目标 1：对于 BF，创建 BF 的动态结构模型。 [BP]G 加合物在插入前、插入和插入后位点的开放二元和封闭三元复合物中，以解释在低温和高温下观察到的堵塞和罕见的旁路具体目标 2：对于 Dpo4，创建 DPO4 的动态结构模型[BP]G 加合物在插入位点的二元和三元复合物中，以解释更容易的旁路、观察到的序列上下文效应和温度效应我们提出的具体目标将提供连接功能与结构的分子细节。