DNA REPAIR IN NORMAL AND MUTATION-PRONE SEQUENCES

DNA会受到许多化学和物理环境因素的破坏。多数 损伤可以通过其中一个细胞DNA修复系统移除，但有些 持续性损伤阻止DNA复制或导致DNA错误 复制，导致突变和功能改变。就是坚持不懈 这种损坏是修复系统随机疏忽的结果或 它反映了受损场地的基本属性吗？在细菌中 大肠杆菌，大多数大而笨重的加合物都是由UVR系统修复的， 包括N-乙酰氧基-N-乙酰基-2-氨基荧烯(-AAF)形成的加合物和 N-羟基-N-2-氨基荧烯(-AF)。然而，间接证据表明 某些易突变序列中的That-AAF加合物很差 修好了。这些序列中加合物的存在可能会诱导 向Z型DNA的局部化转变。 在本项目中，UvrABC蛋白与-AAF加合物的相互作用 我们将检查易突变和正常序列中的And-AF加合物 直接在体外。高分辨率凝胶电泳法将结合 用特定的化学反应和酶反应来定量测定 DNA损伤、UvrA结合和UvrABC切割的顺序分布。 这种方法将允许序列特定的加合物结构相互作用 不寻常地与维修系统一起识别，并将允许 对潜在结构特征的探索。DNA的敏感性 构象转变为变异的性质和浓度 溶液中的盐将被利用来探索 与修复蛋白的相互作用发生了变化。 尽管UVR系统使用DNA螺旋中的扭曲来检测损伤， 只能识别特定形式的失真。损伤诱因 DNA中不寻常的扭曲可能会逃脱修复系统的检测。 由于大肠杆菌的UVR系统与修复系统有共同的元素 这项研究的结果可能会解释 DNA损伤的持续性、DNA修复之间的特定联系 以及诱变或致癌。

DNA is damaged by many chemical and physical environmental agents. Most damage is removed by one of the cellular DNA repair systems, but some persistent damage blocks DNA replication or causes errors in DNA replication, leading to mutations and altered function. Is the persistence of this damage the result of random oversight by the repair systems or does it reflection fundamental properties of the damaged site? In the bacterium Escherichia coli, most large bulky adducts are repaired by the uvr system, including adducts formed by N-acetoxy-N-acetyl-2-aminofluorene (-AAF) and N-hydroxy-N-2-aminofluorene (-AF). However, indirect evidence suggests that -AAF adducts within certain mutation-prone sequences are poorly repaired. The presence of the adduct in these sequences may induce a localized transition to Z-form DNA. In this project, the interaction of the UvrABC proteins with -AAF adducts and -AF adducts in mutation-prone and normal sequences will be examined directly in vitro. High resolution gel electrophoresis will be combined with specific chemical and enzymatic reactions to determine quantitatively the sequence distribution of DNA damage, UvrA binding and UvrABC incision. This method will allow sequence-specific adduct structures that interact unusually with the repair system to be identified and will allow an exploration of the underlying structural features. The sensitivity of DNA conformational transitions to variation in the nature and concentration of salts in the solution will be exploited to probe the physical basis for altered interactions with the repair proteins. Although the Uvr system uses distortion in the DNA helix to detect damage, only a specific form of distortion may be recognized. Damage inducing unusual distortions in the DNA may escape detection by the repair system. Since the Uvr system of Escherichia coli has elements in common with repair systems in higher organisms, the results of this study may illuminate specific connections between the persistence of damage in DNA, DNA repair and mutagenesis or carcinogenesis.