RELATIONSHIP BETWEEN DNA STRUCTURE AND ADDUCT FORMATION

DNA 结构与加合物形成之间的关系

• 批准号: 6352328
• 负责人: BARRY GOLD
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-10 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6352328
• 关键词: DNA; X ray crystallography; adduct; alkylating agents; alkylation; chemical structure function; crosslink; deoxyuridine; fluorescence resonance energy transfer; gene mutation; mechlorethamine; methylguanidine; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleoside analog

The equilibrium binding and covalent interactions of carcinogens and antineoplastic agents with double-stranded DNA involves complex steric and electronic factors that are not well understood. This is due in part to the ability of DNA to adopt different conformations under different conditions. An example of this is the efficient N7-G-to-N7-G interstrand crosslinking of DNA by the nitrogen mustard mechlorethamine at complementary 5'- GNC-3'/3'-CNG-5' sequences (N=any nucleotide). This crosslink is highly deformed because the connector between the two strands is greater than 1.5 Angstrom units too short to accommodate a classical B-DNA structure. We propose that there is a connection between the formation of the unanticipated 5'-GNC crosslink from mechlorethamine, which passes through a cationic monofunctional lesion, and the observed bending of DNA induced by the localization of cationic charge in the major groove by the introduction of 5-(omega-aminoalkyl)uridine residues into DNA. Specifically, it is proposed that the initial monofunctional mustard adduct induces a conformational change, i.e., kink, in DNA that allows the 5'-GNC and N7-G-N3-A crosslinks to form. The Specific Aims are to: (1) Determine the location of the omega- aminoalkyl sidechains and their affect on DNA structure using NMR, crystallography, fluorescence resonance energy transfer, and chemical probes. The impact of a charged purine, i.e., N7- methylguanine, on DNA structure will also be determined. (2) Evaluate how the phasing of cationic sidechains with an A-tract modulates DNA bending as measured by aberrant gel mobility. These phasing experiments will allow us to verify how and to what extent the cationic sidechains distort DNA. (3) Thermodynamically characterize DNA containing charged sidechains, and a N7-methylguanine lesion. The goal is to determine how the cationic charge on the sidechain and/or nucleotide induces changes in solvation that can contribute to DNA bending. (4) Explore the sequence selectivity and kinetics for DNA crosslinking by nitrogen mustards and non-charged analogues. The results from this aim will provide evidence for the role that stable and/or transient cationic charge plays in the sequence selectivity for N7-G to Ny-G interstrand crosslinking. The formation of the putative N7-G to N3-A crosslink by mechlorethamine will be also verified. Completion of these Specific Aims will provide new and fundamental information on: (a) the origin of the sequence specificity of antineoplastic DNA crosslinking agents; (b) how to design efficient DNA crosslinking agents; and (c) how the interaction of DNA with charged species, including basic amino acid sidechains, can induce DNA deformation.

致癌剂和抗肿瘤剂与双链 DNA 的平衡结合和共价相互作用涉及复杂的空间和电子因素，但目前尚不清楚。 这部分是由于 DNA 在不同条件下具有不同构象的能力。 一个例子是氮芥氮芥在互补的 5'-GNC-3'/3'-CNG-5' 序列（N=任何核苷酸）处有效地进行 DNA 的 N7-G 至 N7-G 链间交联。 这种交联高度变形，因为两条链之间的连接器大于 1.5 埃单位，太短而无法容纳经典的 B-DNA 结构。 我们提出，由氮芥形成的意料之外的 5'-GNC 交联（穿过阳离子单功能损伤）与通过引入5-(omega-氨基烷基)尿苷残基进入DNA。具体来说，有人提出，最初的单功能芥子加合物会诱导 DNA 中的构象变化，即扭结，从而允许形成 5'-GNC 和 N7-G-N3-A 交联。 具体目标是： (1) 使用 NMR、晶体学、荧光共振能量转移和化学探针确定 omega-氨烷基侧链的位置及其对 DNA 结构的影响。 带电荷的嘌呤（即 N7-甲基鸟嘌呤）对 DNA 结构的影响也将被确定。 (2) 评估带有 A-tract 的阳离子侧链的定相如何调节 DNA 弯曲（通过异常凝胶迁移率测量）。这些定相实验将使我们能够验证阳离子侧链如何以及在何种程度上扭曲 DNA。 (3) 热力学表征含有带电侧链的 DNA 和 N7-甲基鸟嘌呤损伤。 目标是确定侧链和/或核苷酸上的阳离子电荷如何诱导溶剂化变化，从而导致 DNA 弯曲。 (4) 探索氮芥与不带电荷类似物交联DNA的序列选择性和动力学。 这一目标的结果将为稳定和/或瞬时阳离子电荷在 N7-G 至 Ny-G 链间交联的序列选择性中所起的作用提供证据。 氮芥形成的推定 N7-G 至 N3-A 交联也将得到验证。 完成这些具体目标将提供以下方面的新的基本信息： (a) 抗肿瘤 DNA 交联剂序列特异性的起源； (b) 如何设计高效的DNA交联剂； (c) DNA 与带电物质（包括碱性氨基酸侧链）的相互作用如何引起 DNA 变形。