Benzo(a)pyrene Mutagenic Mechanisms

苯并(a)芘诱变机制

• 批准号: 7531802
• 负责人: EDWARD L LOECHLER
• 金额: $ 44.59万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-30 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7531802
• 关键词: Active Sites; Amino Acids; Benzo(a)pyrene; Bypass; Cells; DNA; DNA-Directed DNA Polymerase; Environmental Carcinogens; Escherichia coli; Goals; Homology Modeling; Human; Image; In Vitro; Lesion; Literature; Modeling; Molecular Conformation; Mutagenesis; Mutation; Pathway interactions; Pattern; Polymerase; Roentgen Rays; Role; Structure; abstracting; adduct; mutant

The potent environmental carcinogen benzo[a]pyrene (B[a]P) is metabolically activated in cells to (+)-anti- B[a]PDE, which forms one primary adduct: [+ta]-B[a]P-N2-dG. [+ta]-B[a]P-N2-dG induces different mutational patterns depending on sequence context (e.g., >95% G->T vs. 95% G->A in 5'-TGC vs. 5'-AGA sequences). Evidence suggests that these different mutations arise from different adduct conformations (as influenced by sequence context), when bypassed by different DNA polymerases. For [+ta]-B[a]P-N2-dG, we showed that E. coli DNA Pol V is involved in dATP bypass (G->T mutations), while dCTP insertion (no mutation) involves Pols IV and V. With the mirror image adduct [-ta]-B[a]P-N2-dG, Pol V does dATP insertion, while Pol IV alone is required for dCTP bypass. Literature findings suggest that in general DNA Pol V has two modes of adduct bypass: (1) correct dNTP insertion, and (2) default dATP insertion. Understanding the mechanism of correct vs. mutagenic insertion is hampered by no X-ray structure for UmuC, which is the the polymerase subunit of DNA Pol V. Using homology modeling, we constructed a UmuC model, which revealed active site amino acids potentially involved in dictating dNTP insertion. Active site amino acids were changed. In cells we showed that mutant-UmuCs could increase (up to ~10-fold), or decrease (~5-fold) dATP insertion compared to wt-UmuC. The goal of this project is to understand what amino acid residues define correct (dCTP) vs. incorrect (dATP) insertion for Pols IV and V and how these pathways are controlled by the cell. Studies in cells and in vitro with mutant and wild type Pols IV and V are proposed. Literature findings show that Pol IV is equivalent to human Pol k, while Pol V is equivalent to human Pol h. Aim 1. Establish the roles of Pol IV vs. Pol V in cells; i.e., which does insertion vs. extension. Aim 2: Determine what amino acids in Pols IV and V control correct (dCTP) vs incorrect (dATP) insertion; e.g., why does Pol IV do correct (dCTP) insertion, while Pol V does incorrect (dATP) insertion, with [-ta]-B[a]P-N2-dG. Aim 3: Determine what lesion-bypass Pols are involved in G->A mutagenesis (dTTP insertion).

强环境致癌物苯并[a]芘（B[a]P）在细胞中被代谢活化， B[a]PDE，其形成一种主要加合物：[+ta]-B[a]P-N2-dG。[+ta]-B[a]P-N2-dG诱导不同的 取决于序列背景的突变模式（例如，5 '-TGC与5'-阿加中> 95% G->T与95% G->A 序列）。有证据表明，这些不同的突变来自不同的加合物构象（如 受序列环境影响）。对于[+ta]-B[a]P-N2-dG，我们 表明E. coli DNA Pol V参与dATP旁路（G->T突变），而dCTP插入（无 在镜像加合物[-ta]-B[a]P-N2-dG的情况下，Pol V使dATP 插入，而dCTP旁路仅需要Pol IV。文献研究结果表明，一般来说，DNA聚合物 V有两种加合物旁路模式：（1）正确的dNTP插入，和（2）默认的dATP插入。 理解正确插入与诱变插入的机制受到没有X射线结构的阻碍， UmuC是DNA Pol V的聚合酶亚基。 UmuC模型，其揭示了可能参与支配dNTP插入的活性位点氨基酸。活性 位点氨基酸发生变化。在细胞中，我们发现mupla-UmuCs可以增加（高达~10倍），或 与wt-UmuC相比，dATP插入减少（约5倍）。 该项目的目标是了解哪些氨基酸残基定义正确（dCTP）与不正确（dATP） 插入Pol IV和V以及这些途径如何由细胞控制。细胞和体外研究 提出了突变型和野生型Pol IV和V。文献结果显示，Pol IV等同于 人Pol k，而Pol V等同于人Pol h。 目标1。确定Pol IV与Pol V在细胞中的作用;即，插入和伸展的对比目标二： 确定Pol IV和V中的哪些氨基酸控制正确（dCTP）与不正确（dATP）插入;例如，为什么 使用[-ta]-B[a]P-N2-dG，Pol IV进行正确的（dCTP）插入，而Pol V进行不正确的（dATP）插入。 目的3：确定哪些病变旁路Pol参与G->A诱变（dTTP插入）。