DYNAMICS OF DNA DAMAGE RECOGNITION BY REPAIR ENZYMES

描述：这个持续项目的长期目标是使用一个联合的 开发分子的理论-实验协同方法 了解特定DNA损伤和修复的原理。在 拟议的新项目期间，胸腺嘧啶二聚体(TD)的两种修复机制将 被研究。一种机制是内切酶V(EndoV)，即 特别识别TD并通过切除修复它。另一种修理 这一过程是通过无差错的跨病变合成完成的。EndoV 特别识别TD并将补充腺嘌呤翻转到 将TD的5‘-胸腺嘧啶放入蛋白质口袋。看来，这一机制 根部翻转是根部切除修复的普遍现象。因此，调查人员将 把这个项目的重点放在了解分子、能量和 区分受损DNA中碱基翻转和DNA损伤中碱基翻转的动力学因素 没有受损的DNA。酶的专一性不仅取决于 识别事件，也受到催化机制的影响。其催化步骤为 EndoV由糖基酶步骤和裂解酶步骤组成。调查人员 建议研究内毒素的酶催化作用机理 对DNA修复中的选择性的贡献。跨损伤合成是一种新的 发现了几种聚合酶的活性。聚合酶ETA(Poleta)是一种 成功合成正确碱基序列的特定示例 在TD对面。Poleta的处理能力很低，这表明它对 DNA与传入的核苷三磷酸(NTP)结合。低处理能力是 参与的其他几种聚合酶(例如聚合酶β)的特性 DNA修复。研究人员提出，该机制的关键特征 Poleta无差错跨病变DNA合成的能力来自于它的能力 识别DNA的特殊结构和动力学性质 TD和酶中优先结合脱氧-ATP的位置 其他核苷酸部分是由蛋白质与 含TD的模板DNA。调查人员建议确定dATP 在与其他低进展性聚合酶同源性的基础上结合部位， 利用分子建模技术构建模型站点并对所建模型进行测试 通过光谱和热力学实验进行的预测。调查人员 将这些目标呈现为完全集成的协作方法，在此方法中 以互补的方式将实验研究和理论研究结合起来。他们觉得 这种综合方法的结果将导致更好的 对影响修复特异性因素的理解 酶，并在发展更一般的理解中发挥重要作用 特定的蛋白质-DNA相互作用。

DESCRIPTION:The long-term goal of this continuing project is to use a combined theoretical-experimental collaborative approach to develop a molecular understanding of the principles of specific DNA damage and repair. In the proposed new project period, two mechanisms of thymine dimer (TD) repair will be studied. One mechanism is that of the enzyme endonuclease V (endoV) that specifically recognizes the TD and repairs it by excision. The other repair process is accomplished by an error-free translesion synthesis. EndoV specifically recognizes the TD and flips the complementary adenine to the 5'-thymine of the TD into a protein pocket. It appears that this mechanism of base flipping is universal to base excision repair. Thus the investigators will focus this project on gaining an understanding of the molecular, energetic and kinetic elements that differentiate base flipping in damaged DNA from that in undamaged DNA. The specificity of enzymes is determined not only by the recognition event, but also by the catalytic mechanism. The catalytic steps of endoV consist of a glycosylase step followed by a lyase step. The investigators propose to study the mechanism of enzymatic catalysis in endoV as a contribution to selectivity in DNA repair. Translesion synthesis is a newly discovered activity of several polymerases. Polymerase eta (poleta) is a particular example that successfully synthesizes the correct base sequence opposite the TD. Poleta has low processivity, suggesting a tolerant site for DNA and the incoming nucleoside triphosphate (NTP) binding. Low processivity is characteristic of several other polymerases (e.g., polymerase beta) engaged in DNA repair. The investigators propose that critical features of the mechanism of error-free translesion DNA synthesis by poleta are derived from its ability to recognize the special structural and dynamic properties of DNA containing a TD and that the site in the enzyme that preferentially binds deoxy-ATP over other nucleotides is in part formed by the interaction of the protein with the TD-containing template DNA. The investigators propose to identify the dATP binding site on the basis of homology with other low-processive polymerases, construct a model site by molecular modeling techniques and test the modeled predictions by spectroscopic and thermodynamic experiments. The investigators present these aims as a fully integrated collaborative approach in which they combine experimental and theoretical studies in a complementary way. They feel that the results of this integrated approach will lead to a better understanding of the factors that contribute to the specificity of repair enzymes and play an important role in developing a more general understanding of specific protein-DNA interactions.