RELATIONSHIP BETWEEN DNA STRUCTURE AND ADDUCT FORMATION

致癌物和抗肿瘤药物与双链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。这方面的一个例子是氮芥氯胺在互补的5'- GNC-3'/3‘- cng -5’序列（N=任何核苷酸）上有效的n7 - g到n7 - g链间交联DNA。这种交联是高度变形的，因为两条链之间的连接器大于1.5埃，太短，无法容纳经典的B-DNA结构。我们提出，甲基三胺通过阳离子单功能损伤形成意想不到的5'- gnc交联，与5-（ω -氨基烷基）尿苷残基引入DNA，导致主凹槽中阳离子电荷定位而引起的DNA弯曲之间存在联系。具体来说，有人提出最初的单功能芥菜加合物在DNA中诱导构象变化，即扭结，从而使5'-GNC和N7-G-N3-A交联形成。具体目的是：(1)利用核磁共振、晶体学、荧光共振能量转移和化学探针确定-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)通过异常凝胶迁移率来评估带有a束的阳离子侧链的相位如何调节DNA弯曲。这些相位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的DNA，以及n7 -甲基鸟嘌呤损伤。目的是确定侧链和/或核苷酸上的阳离子电荷如何在溶剂化中诱导可能导致DNA弯曲的变化。(4)探讨氮芥及其非带电类似物对DNA交联的序列选择性和动力学。该结果将为稳定和/或瞬态阳离子电荷在N7-G到Ny-G链间交联的序列选择性中所起的作用提供证据。氯胺对N7-G到N3-A交联反应的形成也将得到验证。这些特异性目标的完成将为以下方面提供新的基础信息：(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）的相互作用如何诱导DNA变形。

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.