ERROR CORRECTION IN DNA SYNTHESIS

DNA 合成中的错误纠正

• 批准号: 3270462
• 负责人: MYRON GOODMAN
• 金额: $ 17.37万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270462
• 关键词: 5 bromouracil; DNA directed DNA polymerase; Escherichia coli; bacteriophage T4; bioenergetics; chemical bond; conformation; electrophoresis; ionization; microorganism culture; microorganism genetics; molecular genetics; molecular site; mutagens; neoplasm /cancer genetics; nucleic acid sequence; nucleotide analog; phosphodiesterases; radioassay; tautomer; tissue /cell culture

The field of mutagenesis has benefited from recent important technological advances in cloning and sequencing of DNA. These new methods have been instrumental in establishing the nature and location of mistakes made by DNA polymerases. Now that we know what mutagenic events are occurring in vitro, the question is why do they occur in particular locations on DNA and what determines their frequency of occurrence. The overall objective of this study is aimed at elucidating the kinetic mechanisms of DNA polymerase fidelity. Experiments are proposed in which the kinetics of insertion of single nucleotides, both correct and incorrect, can be determined at specific DNA sites. The data will be used to determine to what extent DNA polymerases utilize base pairing free energy differences or active site geometric contraints to favor the formation of Watson-Crick base pairs over non-complementary base pairs. An analysis of the kinetics of base insertion on unaltered DNA templates will be expanded to include the response of polymerase to a biologically significant noncoding lesion on the DNA template, the apurinic/apyrimidinic site. We will determine how the absence of a coding base influences both the rate and concentration dependence for base insertion opposite the lesion and continuation beyond. The kinetics of incorporating modified nucleotides will also be investigated. Studies with modified bases are fundamentally important for understanding both chemical carcinogenesis and cancer chemotherapy, but further, the homologous series of modified bases that we propose to synthesize will be used to determine how systematic changes in base stacking, ionization, tautomerization, and conformation influence kinetics and fidelity. A version of the Sanger dideoxy DNA sequencing method using modified nucleotides will be used as a rapid and sensitive screening assay to reveal the precise identity of base mispairs and to locate potential mutagenic "hot" and "cold" spots over extended regions of DNA. Nucleotide insertion kinetics at hot and cold spots will then be investigated. In a final series of experiments, we propose to measure the effect of 5-bromouracil, a mutagenic base analogue, on intracellular dNTP pool sizes. Perturbation of dNTP pool sizes is an integral component in understanding base analogue-induced mutagenesis in vivo and this study represents an important continuation of work initiated during the previous grant period.

诱变领域受益于最近的重要技术， DNA克隆和测序的进展。 这些新方法已经 有助于确定所犯错误的性质和位置， DNA聚合酶。 既然我们知道了在这些细胞中 在体外，问题是为什么它们发生在DNA上的特定位置， 是什么决定了它们出现的频率 的总体目标 本研究旨在阐明DNA聚合酶的动力学机制 忠诚 实验中提出的动力学插入 单核苷酸，正确和不正确的，可以确定在 特定的DNA位点 这些数据将用于确定DNA在多大程度上 聚合酶利用碱基配对自由能差或活性位点 几何约束有利于沃森-克里克碱基对的形成， 非互补碱基对。 碱的动力学分析 在未改变的DNA模板上的插入将被扩展到包括 聚合酶对生物学显著非编码损伤的反应 DNA模板，脱嘌呤/脱嘧啶位点。 我们将决定如何 编码碱基的缺失影响了速率和浓度 依赖于病变对面的基底插入和继续延伸。 掺入修饰的核苷酸的动力学也将被描述。 研究了 对修饰碱基的研究对于 了解化学致癌作用和癌症化疗，但 此外，我们提出的修饰碱基的同源系列， 将采用综合方法来确定碱系统性变化 堆积、电离、互变异构和构象影响动力学 和忠诚 桑格双脱氧DNA测序方法的一个版本， 修饰的核苷酸将被用作快速和灵敏的筛选测定， 来揭示碱基错配的精确身份并定位潜在的 在DNA延伸区域上的诱变"热点"和"冷点"。 核苷酸 然后将研究在热点和冷点的插入动力学。 中 最后一系列的实验，我们建议测量的效果， 5-溴尿嘧啶（一种致突变碱基类似物）对细胞内dNTP池的影响 尺寸. dNTP池大小的扰动是 了解碱基类似物诱导的体内诱变和本研究 这是前一阶段工作的重要延续， 补助期。