D(GPG) IN CRUCIFORM LOOPS AS TARGETS FOR CISPLATIN

这是香农奖，为这项研究提供部分支持 项目低于指定研究所的资助范围，但 都在最优秀的边缘。香农奖旨在提供 支持测试该方法的可行性；开发进一步的测试 改进研究方法；对可用的数据进行二次分析 数据集；或执行可演示PI的离散项目 研究能力或导致额外的重量已经有价值的 申请。以下应用程序取自原始文档 由首席调查员提交。 在这里，我们提出扭曲的DNA结构，特别是环 可能使某些d(GPG)二核苷酸更敏感 作为抗肿瘤药物顺式修饰的靶点- 二氨基二氯铂(cis-DDP或顺铂)。这是一个激进的 偏离cis-DDP绑定到d(GPG)的标准范式 线性DNA中的二核苷酸，随后扭曲DNA双链。我们的 模型是从初步研究得出的，该模型表明顺式顺铂，但 不是临床上不活跃的反式异构体，可以改变动力 双链DNA和十字形之间的平衡以及最近的核磁共振 扁平DNA发夹的结构。这些结构性因素的作用 微扰可能会通过以下方式为药物提供更高的特异性 建立更加顺应环境的立体化学环境 交联物的结构。我们估计， 人类基因组中位于十字形形成序列中的D(GPG)位点 (约5,000)与细胞中发现的药物分子数量相似 在治疗期间(约1,000人)。识别铂的蛋白质 DNA中的加合物(特别是高流动性基团或HMG类 蛋白质)实际上可能识别顺式顺铂稳定的十字形。 这些蛋白质已被证明与合成的四向连接结合， 它们在十字架的底部形成。 在本申请中，我们建议进行研究，以提供进一步的支持 这个新型号。我们建议：i)确定d(GPG)站点是否在 十字环易受顺式顺式二磷酸结合的影响 与线性双链DNA中的相同位点进行比较 线形与挤压十字形铂在d(GPG)位的反应 DNA；ii)确定顺式顺铂有助于稳定 挤压十字形拓扑异构体的二维凝胶分析 含有质粒的十字形；以及iii)确定HMG-I-a 在某些癌细胞中升高的蛋白质-识别和 通过比较与顺式顺铂稳定的十字形的结合 十字形和线形DNA中的蛋白质到d(GPG)位点。

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Here, we propose that distorted DNA structures, specifically the loops of cruciforms, may render certain d(GpG) dinucleotides more susceptible as targets for modification by the antitumor drug cis- diamminedichloroplatinum (II) (cis-DDP, or cisplatin). This is a radical departure from the standard paradigm that cis-DDP binds to d(GpG) dinucleotides in linear DNA and subsequently distorts the DNA duplex. Our model was derived from preliminary studies which show that cis-DDP, but not its clinically inactive trans-isomer, can shift the dynamic equilibrium between duplex DNA and a cruciform, and a recent NMR structure of a platinated DNA hairpin. The role of these structural perturbations may he to provide greater specificity to the drug by setting up the stereochemical environment that is more amenable to the structure of the cross-linked adduct. We estimate that the number of d(GpG) sites in a human genome that lie in cruciform forming sequences (about 5,000) is similar to the number of drug molecules found in a cell during treatment (about 1,000). The proteins that recognize platinum adducts in DNA (particularly the high mobility group or HMG classes of proteins) may actually be recognizing a cis-DDP stabilized cruciforms. These proteins have been shown to bind to synthetic four-way junctions, which form at the base of cruciforms. In this application, we propose studies to provide further support of this new model. We propose to: i) determine whether d(GpG) sites in cruciform loops are significantly more susceptible to cis-DDP binding compared to the same sites in linear duplex DNA by comparing the rate of platinum reaction at a d(GpG) site in linear versus extruded cruciform DNAs; ii) determine the degree to which cis-DDP helps to stabilize the extruded cruciform by two-dimensional gel analysis of topoisomers of cruciform containing plasmids; and iii) determine whether HMG-I--a protein which is elevated in certain cancerous cells--recognizes and binds to cis-DDP stabilized cruciforms by comparing the binding of this protein to d(GpG) sites in cruciform and linear DNAs.