STRUCTURE, PROTEIN RECOGNITION AND ENERGETICS OF DAMAGED DNA

我们工作的总目标是确立结构和功能 突变与DNA修复的关系及发展计算 可用于预测修复酶的模式的方法 识别受损的DNA。我们建议确定解的结构 几个含有诱变加合物的DNA分子，锌指基序 FpG蛋白及其与DNA的1：1复合体。有待研究的加合物 包括乙酰氨基-C(8)和N_2-鸟氨酸、氨基-F-G(8)- 鸟嘌呤，苯并咪唑并吡啶-C(8)-鸟嘌呤，8-氧基鸟嘌呤，8-氧基腺嘌呤， 8-氨基鸟嘌呤，以及碱性位点。基于诱变研究 在(项目2)中，这些加合物将被结合到DNA中 双链作为受损DNA的模型；错位凸起的双链，作为模型 用于移码突变；以及作为模型的引物模板 复制分叉中间体。这些加合物将在 约翰逊博士的实验室(项目I)；几个基地将 同位素标记以提高核磁共振分辨率。一维和二维 高分辨率核磁共振实验将在加成的DNA上进行 分子，使用石溪的600兆赫分光光度计。核磁共振数据 将用于推导二面体约束、距离约束和体积约束， 当结合到分子力学和动力学计算中时， 将建立上述加合物的溶液结构。 计算将在我们的Silicon Graphics计算机上运行，以及 国家科学基金会的超级计算机。我们建议将我们的计算方法扩展到 基于氢键模式识别的大分子对接 包括动态灵活性和水桥连接到我们目前僵硬的- 身体分子模型。我们的计算机程序的增强将被编码 并在应用中心的超立方体并行计算机设施上运行 数学系。

The overall goal of our work is to establish structure-function relationships in mutagenesis and DNA repair and to develop computational methods that can be used to predict patterns by which repair enzymes recognize damaged DNA. We propose to determine solution structures of several DNA molecules containing mutagenic adducts, the zinc finger motif of Fpg protein, and its 1:1 complex with DNA. Adducts to be studied include acetylaminofluorene-C(8)- and N2-guanine, aminofluorene-G(8)- guanine, phenilimidazopyridine-C(8)-guanine, 8-oxoguanine, 8-oxoadenine, 8-aminoguanine, as well as abasic sites. Based on mutagenesis studies conducted in (Project 2), these adducts will be incorporated into DNA duplexes as models for damaged DNA; misaligned bulged duplexes, as models for frameshift mutagenesis; and primer-templates as models for replication fork intermediates. These adducts will be synthesized in the laboratories of Dr. Johnson (Project i); several bases will be isotopically labeled to enhance NMR resolution. One- and two-dimensional high resolution NMR experiments will be performed on the adducted DNA molecules, using the 600 MHz spectrophotometer at Stony Brook. NMR data will be used to derive dihedral-, distance- and volume-restraints which, when incorporated into molecular mechanics and dynamics calculations, will establish the solution structure of the above-mentioned adducts. Calculations will run on our Silicon Graphics computers, as well as on NSF supercomputers. We propose to expand our computational methods for macromolecular docking based on hydrogen bond pattern recognition by including dynamic flexibility and water bridging to our current, rigid- body molecular model. Enhancements of our computer programs will be coded and run on the hypercube parallel computer facility at the Applied Mathematics Department.