MOLECULAR PHARMACOLOGY OF TUMOR AND VIRUS INHIBITORS

这项研究的重点是DNA损伤的生物学后果， 阐明致癌过程中主要事件的总体目标。 一 主要的主题是建立结构之间的关系 受损的DNA和参与DNA复制的酶的功能， 修复. 为此，已经开发了新的实验系统。 位点特异性诱变涉及一种策略，其中穿梭质粒 载体，包含一个单一的定义病变，允许复制， 哺乳动物细胞或细菌。 诱导突变的位置是 通过DNA序列分析确定。 引物延伸反应， 由DNA聚合酶催化，结合稳态动力学分析， 被用来探索translesional合成和诱变事件， 体外 我们的具体目标是（a）建立移码突变模型 在错位的DNA模板和动力学方面， 合成;（B）阐明序列背后的分子基础 背景对碱基替换和缺失的影响;（c）了解 SOS功能在跨病灶合成中的作用;（d）发现 双链修复过程中产生突变的途径 DNA中的无碱基位点;（e）证明DNA之间的差异 聚合酶在其产生由DNA引起的突变的能力方面具有优势， （f）开发预测致突变特异性的体外试验 （g）探索DNA损伤的机制， 损伤增加了哺乳动物中同源重组的频率 细胞和细菌;以及（h）建立以下的溶液结构： 在缺失诱变过程中形成的未对齐中间体。 设计了额外的研究（a）以确定底物 Fpg蛋白的特异性;（B）确定N末端的作用 （c）在该酶的催化功能中;（c）揭示结构 锌指结构域氧化损伤DNA结合的基础;（d） 阐明DNA糖基化活性的催化机制;（e） 检测Fpg蛋白及其底物上的官能团， 促进氧化损伤的“识别”;（f）建立 Fpg蛋白与腺嘌呤DNA形成复合物的结构 糖基化物及其DNA底物的类似物;以及（g）研究 底物结合和所选AP核酸内切酶的作用机制， 和（h）定量Fpg蛋白对DNA修复的贡献， 细胞

This research focusses on the biological consequences of DNA damage with the overall goal of elucidating primary events in carcinogenesis. A principal theme is to establish relationships between the structure of damaged DNA and the functions of enzymes involved in DNA replication and repair. Novel experimental systems have been developed for this purpose. Site specific mutagenesis involves a strategy in which a shuttle plasmid vector, containing a single defined lesion, is allowed to replicate in mammalian cells or bacteria. The position of mutations induced is established by DNA sequence analysis. Primer-extension reactions, catalyzed by DNA polymerase, coupled with steady-state kinetic analysis, are used to explore translesional synthesis and mutagenic events in vitro. Our specific aims are (a) to establish models for frameshift mutagenesis in terms of misaligned DNA templates and kinetics governing translesional synthesis; (b) to elucidate the molecular basis underlying sequence context effects on base substitutions and deletions; (c) to understand the role of SOS functions in translesional synthesis; (d) to discover pathways by which mutations are generated during repair of bistrand abasic sites in DNA; (e) to demonstrates differences between DNA polymerases in their abilities to generate mutations arising from DNA damage; (f) to develop in vitro assays that predict mutagenic specificity for defined DNA lesions in vivo; (g) to explore mechanisms by which DNA damage enhances the frequency of homologous recombination in mammalian cells and bacteria; and (h) to establish the solution structure of misaligned intermediates formed during deletion mutagenesis. Additional studies are designed (a) to determined the substrate specificity of Fpg protein; (b) to establish the role of the N-terminus in the catalytic function of this enzyme; (c) to reveal the structural basis for binding of the zinc finger domain oxidatively-damaged DNA; (d) to elucidate a catalytic mechanism for DNA glycosylate activity; (e) to detect functional groups on Fpg protein and its substrates that facilitate "recognition" of oxidative damage; (f) to establish the structure of complexes formed between Fpg protein or adenine DNA glycosylate and analogs of their DNA substrates; and (g) to study substrate binding and mechanism of action of selected AP endonucleases, and (h) to quantify the contribution of Fpg protein to DNA repair in cells.