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: 7723185
• 负责人: Suse Broyde
• 金额: $ 0.05万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723185
• 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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目前的证据表明，DNA聚合酶通过聚合酶开关模型绕过大体积致癌物-DNA加合物[1-5]。在该模型中，复制型DNA聚合酶以高保真度和效率进行DNA复制，直到它们遇到DNA中的致癌物损伤位点。然而，它们经常被这种病变阻断。在复制性DNA聚合酶从复制叉解离后，可以调用旁路聚合酶来复制通过损伤。在非常低的频率下，复制型DNA聚合酶本身也可以绕过病变[5-9]，大概是如果加合物处于某些允许的构象。通过两种类型的聚合酶的病变旁路可以是致突变的或非致突变的。如果错配的配偶体在加合物的对面掺入，或者当聚合酶试图通过病变时发生引物链相对于模板的滑动，则将发生突变。这些突变，如果发生在某些关键基因，如癌基因或肿瘤抑制基因，可能导致癌症的发生[10]。多环芳烃（PAHs）是有机物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各种物质中，如汽车尾气，烟草烟雾和烤肉和鱼[11-13]。它可以代谢活化为许多代谢物，包括（+）-抗BPDE（苯并[a]芘二醇环氧化物），其在哺乳动物系统中具有高度致突变性和致瘤性[14，15]。代谢物可攻击DNA，主要攻击的碱基是鸟嘌呤;主要形成10 S（+）-反式-抗-[BP]-dG（[BP]G）加合物[16-19]。实验研究[20]已经证明，10 S（+）-反式-抗-[BP]-dG加合物主要阻断细菌复制性DNA聚合酶，芽孢杆菌片段（BF）[21]，具有非常小的旁路。然而，这种相同的加合物更容易被古细菌旁路DNA聚合酶Dpo 4绕过，Dpo 4是DinB家族的成员，也在人类中发现[22]。此外，碱基序列背景（CG*G vs. TG*G，G*= 10 S（+）-trans-anti-[BP]-dG）已显示影响Dpo 4的旁路效率[20]。碱基序列背景对致突变性的影响对于理解不同序列背景下惊人的不同致突变结果是重要的。此外，在BF和Dpo 4的情况下，与37 C相比，在55 C下观察到更大的旁路效率[20]。研究高温是因为BF和Dpo 4都是嗜热酶，其效率在较高温度下最大。我们假设，观察到的不同的复制活动BF和DPO 4的这种加合物是由于它们之间的结构差异，特别是在活性位点，和高温增强聚合酶的灵活性，从而使不太有利的构象的加合物更容易。为了研究导致两种酶在复制BP修饰的DNA中的不同活性的结构因素，我们将进行以下研究，以追求两个具体目标：具体目标1：对于BF，在插入前、插入和插入后位点，创建[BP]G加合物在开放二元和封闭三元复合物中的动态结构模型，解释在低温和高温下观察到的堵塞和罕见的旁路。对于Dpo 4，在插入位点的二元和三元复合物中创建[BP]G加合物的动态结构模型，以解释更容易的旁路，观察到的序列背景效应，我们提出的具体目标将提供连接功能与结构的分子细节。