Sequence Effects of AF-modified DNA Structures

AF 修饰 DNA 结构的序列效应

• 批准号: 6678377
• 负责人: Bongsup P Cho
• 金额: $ 28.43万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678377
• 关键词: DNA repair; DNA replication origin; adduct; circular dichroism; conformation; fluorine; molecular oncology; nuclear magnetic resonance spectroscopy; nucleic acid structure; site directed mutagenesis; stable isotope

DESCRIPTION (provided by applicant): Structural elucidation of DNA adducts is crucial to understanding the initiation of carcinogenesis and ultimately in designing preventative strategies. Arylamine carcinogens are implicated in the etiology of various human cancers. We hypothesize that: (a) arylamine-adducts in DNA exist in two prototype conformations, a base-displaced "stacked" (S) and an external binding normal "B-type" 03); (b) a delicate S/B conformeric balance is modulated by both the base sequence surrounding the adduct site and the nature of specific interactions in the active sites of replication and repair enzymes; and (c) the resulting conformational heterogeneity plays a critical role in determining the mutagenic outcomes. We will use the well-documented aminofluorene (AF)-induced S/B heterogeneity to correlate their propensity to undergo repair and replication. This will be accomplished by the use of 19FNMR coupled with fluorine-labeled AF-modified DNA in various sequence contexts, with or without the presence of a polymerase. Specific aims are to (1) measure the S/B ratios of double or single/double stranded duplexes in various sequence contexts; (2) to measure the S/B ratios of deletion duplexes, whose 3'-next nearest flanking sequence to the lesion is altered; (3) to measure the S/B ratios of 18/9-mer template-primers, whose 5'-flanking sequences are varied, in the presence of the polymerase Klenow Fragment (KFexo-); and (4) to conduct site-specific mutagenesis of the sequences used in Aims 1 and 3. Circular dichroism and melting experiments will also be conducted to define further the adduct structure. The polymerase experiments proposed in Aim 3, in particular, examine the adduct conformation at the replication fork within the active site of a polymerase and may provide,for the first time, a systematic basis for conducting conformation- and adduct-specific mutagenesis in a simulated biological environment. The proposed research will introduce 19F NMR as a powerful structural biology tool in investigating the mechanisms of arylamine mutagenesis.

描述（由申请人提供）：DNA 加合物的结构阐明对于理解致癌作用的起始以及最终设计预防策略至关重要。芳胺致癌物与多种人类癌症的病因有关。我们假设：（a）DNA中的芳胺加合物以两种原型构象存在，碱基置换的“堆叠”（S）和外部结合正常的“B型”03）； (b) 微妙的S/B构象平衡受到加合物位点周围的碱基序列以及复制和修复酶活性位点中特定相互作用的性质的调节； (c) 由此产生的构象异质性在决定诱变结果方面发挥着关键作用。我们将使用有据可查的氨基芴 (AF) 诱导的 S/B 异质性来关联它们进行修复和复制的倾向。这将通过在各种序列环境中使用 19FNMR 与氟标记的 AF 修饰的 DNA 偶联来实现，无论是否存在聚合酶。具体目标是 (1) 测量各种序列背景下双链或单/双链双链体的 S/B 比率； (2) 测量删除双链体的 S/B 比，其 3'-下一个最接近病变的侧翼序列被改变； (3) 在聚合酶 Klenow 片段 (KFexo-) 存在下测量 18/9 聚体模板引物的 S/B 比，其 5'-侧翼序列不同； (4)对目标1和3中使用的序列进行位点特异性诱变。还将进行圆二色性和熔解实验以进一步确定加合物结构。目标 3 中提出的聚合酶实验，特别是检查聚合酶活性位点内复制叉处的加合物构象，并可能首次为在模拟中进行构象和加合物特异性诱变提供系统基础。生物环境。拟议的研究将引入 19F NMR 作为研究芳基胺诱变机制的强大结构生物学工具。