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RELATIONSHIP BETWEEN DNA STRUCTURE AND ADDUCT FORMATION

DNA 结构与加合物形成之间的关系

基本信息

批准号：
6686443
负责人：
BARRY GOLD
金额：
$ 27.5万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-10 至 2005-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6686443
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

项目摘要

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.

致癌物和致癌剂与双链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，这些因素尚未得到很好的理解。这部分是由于DNA在不同条件下采用不同构象的能力。这方面的一个实例是通过氮芥氮芥在互补的5 '-GNC-3'/3 '-CNG-5'序列（N=任何核苷酸）处的DNA的有效N7-G至N7-G链间交联。这种交联是高度变形的，因为两条链之间的连接体大于1.5埃单位，太短而不能容纳经典的B-DNA结构。我们提出，从氮芥形成的未预料到的5 '-GNC交联，它通过阳离子单功能损伤，和观察到的弯曲的DNA诱导的阳离子电荷的本地化通过5-（ω-氨基烷基）尿苷残基引入到DNA的大沟之间的连接。具体地，提出初始单官能芥子气加合物诱导构象变化，即，DNA中允许5 '-GNC和N7-G-N3-A交联形成的扭结。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ω-氨基烷基侧链的位置及其对DNA结构的影响。带电嘌呤，即，N7-甲基鸟嘌呤对DNA结构的影响也将被确定。 (2)评价阳离子侧链与A道的定相如何调节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多大程度上扭曲DNA。 (3)热力学表征含有带电侧链和N7-甲基鸟嘌呤损伤的DNA。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作用的变化，从而导致DNA弯曲。 (4)探索氮分子和不带电类似物对DNA交联的序列选择性和动力学。从这个目标的结果将提供证据的作用，稳定和/或瞬态阳离子电荷在N7-G到Ny-G链间交联的序列选择性。还将验证氮芥形成推定的N7-G至N3-A交联。完成这些特定目标将提供新的和基本的信息：（a）DNA交联剂的序列特异性的起源;（B）如何设计有效的DNA交联剂;和（c）DNA与带电物质（包括碱性氨基酸侧链）的相互作用如何诱导DNA变形。