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; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 当前的证据表明，笨重的致癌-DNA加合物通过聚合酶开关模型被DNA聚合酶绕过[1-5]。在此模型中，复制性DNA聚合酶以高保真和效率进行DNA复制，直到它们符合DNA中的致癌部位。但是，它们经常被这种病变阻止。在复制性DNA聚合酶与复制叉分离后，可以召集旁路聚合酶以复制过后病变。在非常低的频率下，复制性DNA聚合酶本身也可以绕过病变[5-9]，大概是在某些允许构象中加合物的话。两种类型的聚合酶的病变旁路可以是诱变或非毒素。如果将不匹配的伴侣纳入与模板相对于模板的加合物或滑点相对，则在聚合酶试图转移病变时发生突变。如果发生在某些关键基因（例如癌基因或肿瘤抑制基因）中，则这种突变会导致癌症的启动[10]。多环芳烃（PAHS）是在有机材料燃烧过程中产生的环境促抗癌症。苯并[A] pyrene（BP）是最广泛研究的PAH之一，通常在人类摄入或吸入的各种物质中，例如汽车排气，烟草烟雾，烤肉和鱼类[11-13]。可以将其代谢激活为多种代谢产物，包括（+） - 抗BPDE（苯并[A] pyrene二醇环氧），该代谢物在哺乳动物系统中是高度诱变和肿瘤性的[14，15]。这些代谢物可以攻击DNA，而主要攻击的碱是鸟嘌呤。 A 10s（+） - trans-anti- [bp] -dg（[bp] g）加合物主要形成[16-19]。实验研究[20]表明，10s（+） - 跨抗 - [bp] -dg加合物主要阻止细菌复制性DNA聚合酶，芽孢杆菌片段（BF）[21]，旁路很少。但是，通过古细胞旁路DNA聚合酶DPO4更容易绕开相同的加合物，DPO4是在人类中也发现的Dinb家族的成员[22]。此外，已经证明基本序列上下文（CG*G vs. Tg*g*= 10s（+） - trans-anti- [bp] -dg）会影响DPO4的旁路效率[20]。基本序列上下文对诱变性的影响与理解不同序列上下文中令人惊讶的不同诱变结果有关。此外，在BF和DPO4的情况下，与37C相比，在55C时观察到更高的旁路效率[20]。由于BF和DPO4都是嗜热酶，因此研究了高温，其在较高温度下的效率最高。我们假设观察到的BF和DPO4的不同复制活性是由于它们之间的结构差异所致，尤其是在活性位点，并且高温增强了聚合酶的灵活性，从而使加合物的偏爱构型较少。 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具体目标2：对于DPO4，在插入位点创建[BP] G加合成物的动态结构模型，以解释易于旁路，观察到的序列上下文效应以及我们提出的特定目的的温度效应将提供与结构相关的分子细节。