Error-Prone Replicative Bypass of Platinum Adducts

铂加合物的容易出错的复制旁路

• 批准号: 6862628
• 负责人: STEPHEN G CHANEY
• 金额: $ 29.51万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6862628
• 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; protein purification

DESCRIPTION (provided by applicant): Of the platinum complexes currently utilized in cancer chemotherapy oxaliplatin (OX) appears to be less mutagenic than cisplatin (CP) or carboplatin. The mechanism(s) for these differences is not clear, but their mutagenicity is likely to be determined in part by DNA polymerase(s) that catalyze translesion synthesis past Pt-DNA adducts and/or damage-recognition proteins that bind to the adducts and prevent translesion synthesis. We postulate that there are differences in conformation and/or conformational flexibility between CP and OX adducts that affect the efficiency and fidelity of translesion synthesis. We have recently obtained the NMR solution structure for the OX-GG adduct in the mutagenic AGG sequence context, and it is significantly different from all previous solution structures of the CP-GG adduct. Thus, we plan to solve the NMR solution structure of the CP adduct and conduct NMR relaxation studies on OX and CP adducts in the AGG sequence context (aim 1). Our in vitro studies show that pol eta, pol beta, and pol mu are the only DNA polymerases with a significant capacity to bypass CP and OX adducts. Our data show that pol eta expression decreases the mutagenicity of CP, and previous studies have shown that pol beta overexpression increases the mutagenicity of CP. Pol beta is an excellent model for studying translesion synthesis because of the wealth of available structural and kinetic information. HMG-domain proteins such as SRY bind to CP and OX adducts with different affinities and can block translesion synthesis. Thus, we plan to extract NMR structural and dynamic information for OX and CP adducts in complex with pol beta and the damage-recognition protein SRY (aim 2); utilize molecular dynamics to characterize the impact of CP and OX adducts on DNA flexibility and to characterize the interaction of these adducts with pol beta (aim 3); and characterize the pre-steady state kinetics of pol beta with templates containing OX and CP adducts (aim 4). These data will also be used to verify and refine the molecular dynamic simulations performed in aim 3. These experiments will help define the mechanism(s) of Pt drug-induced mutagenesis and will result in models of Pt-DNA-polymerase interactions that will be useful in clarifying the mechanisms of translesion synthesis past bulky DNA adducts.

描述（由申请人提供）：目前用于癌症化疗的铂络合物中，奥沙利铂（OX）似乎比顺铂（CP）或卡铂的致突变性更小。这些差异的机制尚不清楚，但它们的致突变性可能部分是由催化跨损伤合成经过 Pt-DNA 加合物和/或与加合物结合的损伤识别蛋白的 DNA 聚合酶决定的。防止跨损伤合成。我们假设 CP 和 OX 加合物之间的构象和/或构象灵活性存在差异，这会影响跨损伤合成的效率和保真度。我们最近获得了诱变 AGG 序列背景下 OX-GG 加合物的 NMR 溶液结构，它与之前所有 CP-GG 加合物的溶液结构显着不同。因此，我们计划求解 CP 加合物的 NMR 溶液结构，并对 AGG 序列背景下的 OX 和 CP 加合物进行 NMR 弛豫研究（目标 1）。我们的体外研究表明，pol eta、pol beta 和 pol mu 是唯一具有绕过 CP 和 OX 加合物的显着能力的 DNA 聚合酶。我们的数据显示pol eta表达降低了CP的致突变性，之前的研究表明pol beta过度表达增加了CP的致突变性。由于丰富的可用结构和动力学信息，Pol beta 是研究跨损伤合成的优秀模型。 HMG 结构域蛋白（例如 SRY）以不同的亲和力与 CP 和 OX 加合物结合，并且可以阻断跨损伤合成。因此，我们计划提取与 pol beta 和损伤识别蛋白 SRY 复合的 OX 和 CP 加合物的 NMR 结构和动态信息（目标 2）；利用分子动力学来表征 CP 和 OX 加合物对 DNA 灵活性的影响，并表征这些加合物与 pol beta 的相互作用（目标 3）；并用含有 OX 和 CP 加合物的模板表征 pol beta 的前稳态动力学（目标 4）。这些数据还将用于验证和完善目标 3 中进行的分子动力学模拟。这些实验将有助于定义 Pt 药物诱导诱变的机制，并将产生 Pt-DNA-聚合酶相互作用模型，该模型将在有助于阐明跨损伤合成经过大体积 DNA 加合物的机制。