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: 7601434
• 负责人: Suse Broyde
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601434
• 关键词: Active Sites; Affect; Aromatic Polycyclic Hydrocarbons; Automobile Exhaust; Bacillus (bacterium); Base Sequence; Benzo(a)pyrene; 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; Glycol; Grant; Guanine; High temperature of physical object; Human; Institution; Lead; Lesion; Malignant Neoplasms; Meat; Modeling; Molecular; Molecular Conformation; Mutation; Numbers; Oncogenes; Outcome; Pliability; Polymerase; Range; 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; conformer; 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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 目前的证据表明，DNA聚合酶通过聚合酶开关模型绕过了体积庞大的致癌物-DNA加合物[1-5]。在这个模型中，复制的DNA聚合酶以高保真和高效的方式进行DNA复制，直到它们到达DNA中的致癌物损伤部位。然而，它们经常被这样的损伤所阻断。在复制DNA聚合酶与复制叉解离后，可以调用一个旁路聚合酶来复制通过病变。在很低的频率下，复制的DNA聚合酶本身也可以绕过病变[5-9]，假设加合物是某些允许的构象。这两种聚合酶的病变旁路可以是诱变的，也可以是非诱变的。当聚合酶试图转移病变时，如果不匹配的配对被掺入与模板相反的加成物或引物链相对于模板的滑动，就会发生突变。这种突变如果发生在某些关键基因，如癌基因或肿瘤抑制基因，可能会导致癌症的发生[10]。多环芳烃(PAHs)是有机物质燃烧过程中产生的环境致癌物质。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各种物质中，如汽车尾气、烟草烟雾、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)，这在哺乳动物系统中是高度诱变和致癌的[14，15]。代谢产物可以攻击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，同样的加合物更容易被古生菌旁路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，碱基序列环境(CG*G与TG*G，G*=10S(+)-反式抗[BP]-DG)已被证明影响DPO4[20]的旁路效率。碱基序列上下文对突变性的影响对于理解不同序列上下文中令人惊讶的不同突变结果是重要的。此外，在BF和DPO4的情况下，与37C相比，55C时观察到更高的旁路效率[20]。高温的研究是因为BF和DPO4都是嗜热酶，在较高的温度下效率最高。我们假设，观察到的BF和DPO4对该加合物的不同复制活性是由于它们之间的结构差异，特别是在活性部位，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更容易获得。为了研究导致两种酶在复制BP修饰的DNA过程中活性不同的结构因素，我们将开展以下两个特定目标的研究：具体目标1：对于BF，在插入前、插入和插入后，建立开放的二元和闭合的三元复合体中[BP]G加合物的动态结构模型，解释观察到的低温和高温下的堵塞和罕见的旁路。特定目标2：对于DPO4，建立插入位点的二元和三元络合物中的[BP]G加合物的动态结构模型，以解释更容易的旁路、观察到的序列上下文效应，而我们提出的特定目标的温度效应将提供将功能与结构联系起来的分子细节。