ERROR PRONE REPLICATIVE BYPASS OF PLATINUM ADDUCTS

铂加合物的复制旁路容易出错

• 批准号: 6196104
• 负责人: STEPHEN G CHANEY
• 金额: $ 25.47万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6196104
• 关键词: DNA directed DNA polymerase; DNA replication; active sites; adduct; cell line; chemical models; chemical structure function; cis platinum compound; computer simulation; conformation; enzyme activity; gene mutation; molecular dynamics; nuclear magnetic resonance spectroscopy; nucleic acid sequence

Cisplatin is a probable human carcinogen. Platinum compounds such as oxaliplatin have shown promise for the treatment of cisplatin resistant tumors, but their potential mutagenicity is unknown. The mutagenicity of these compounds is likely to be determined in part by the DNA polymerase(s) which catalyze error-prone translesion synthesis past Pt-DNA adducts. The identity if these polymersases is not currently known, but pol beta, pol zetu, and pol eta have all been shown to perform translesion synthesis past Pt-DNA adducts in vitro. Cell lines exist which under- or over-express each of these polymersases. Thus, we plan to determine the effects of pol beta, pol zetu, and pol eta expression on the mutagenicity and cytotoxicity of cisplatin and oxaliplatin adducts (Aim 1). An analysis of the conformational differences of the adducts formed by these compounds is likely to be essential for understanding the mechanisms which determine their mutagenicity and efficacy. Thus, we plan to use a combination of NMR and molecular modeling to characterize the adducts formed by these compounds (Aim 2). Detailed information on the efficiency and fidelity of translesion synthesis by purified DNA polymerases is important for confirming the contribution of those polymersases to error-prone translesion synthesis in vivo (Aim 1) and for modeling their interaction with the adducts (Aim 4). Thus, the efficiency and fidelity of translesion synthesis past cisplatin and oxaliplatin adducts by pol beta, pol zetu, and/or pol eta will be determined in vitro (Aim 3). It is likely that the conformation and mutagenicity of Pt-DNA are affected by their binding to the active site of the polymerases involved in error-prone translesion synthesis. Based on the conformational data on the adducts (Aim 2) and the known crystal structures of pol beta, molecular dynamics simulations will be used to model the interactions of pol beta with each of the adducts (Aim 4). This project will define the role of pol beta, pol zetu, and pol eta in error-prone replicative bypass of Pt-DNA adducts. It will also result in models of Pt-DNA adduct conformation and polymerase-adduct interaction that will be useful in understanding the mechanism(s) of mutagenesis by these and other Pt complexes.

顺铂是一种可能的人类致癌物。 奥沙利铂等铂化合物已显示出治疗顺铂耐药肿瘤的前景，但其潜在的致突变性尚不清楚。 这些化合物的致突变性可能部分是由 DNA 聚合酶决定的，DNA 聚合酶催化 Pt-DNA 加合物的易错跨损伤合成。 目前尚不清楚这些聚合酶的身份，但 pol beta、pol zetu 和 pol eta 均已显示可在体外通过 Pt-DNA 加合物进行跨损伤合成。 存在这些聚合酶表达不足或过度表达的细胞系。因此，我们计划确定 pol beta、pol zetu 和 pol eta 表达对顺铂和奥沙利铂加合物的致突变性和细胞毒性的影响（目标 1）。 对这些化合物形成的加合物的构象差异的分析可能对于理解决定其致突变性和功效的机制至关重要。因此，我们计划结合使用 NMR 和分子建模来表征这些化合物形成的加合物（目标 2）。关于纯化 DNA 聚合酶跨损伤合成的效率和保真度的详细信息对于确认这些聚合酶对体内易错跨损伤合成的贡献（目标 1）以及模拟它们与加合物的相互作用（目标 4）非常重要。 因此，将在体外确定 pol beta、pol zetu 和/或 pol eta 跨损伤合成顺铂和奥沙利铂加合物的效率和保真度（目标 3）。 Pt-DNA 的构象和致突变性很可能受到其与参与易错跨损伤合成的聚合酶活性位点的结合的影响。 基于加合物的构象数据（目标 2）和已知的 pol beta 晶体结构，分子动力学模拟将用于模拟 pol beta 与每种加合物的相互作用（目标 4）。该项目将定义 pol beta、pol zetu 和 pol eta 在容易出错的 Pt-DNA 加合物复制旁路中的作用。 它还将产生 Pt-DNA 加合物构象和聚合酶-加合物相互作用的模型，这将有助于理解这些和其他 Pt 复合物的诱变机制。