Origins of differential response to DNA lesions by DNA polymerases: Insights throuth directed enzyme evolution

DNA 聚合酶对 DNA 损伤的差异反应的起源：通过定向酶进化的见解

• 批准号: 5427221
• 负责人: Professor Dr. Andreas Marx
• 金额: --
• 依托单位: Arbeitsgruppe Organische Chemie / Zelluläre Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5427221?language=en
• 关键词: Origins; differential; response; DNA; lesions

Environmental stress such as ultraviolet light damages DNA, for example by forming pyrimidine dimers. It has long been obscure how cells perform DNA synthesis past these lesions. Only recently, novel specialized DNA polymerases were identified to be involved in translesion synthesis. These enzymes are found in a variety of organisms and e.g. are involved in the suppression of skin cancer in humans. Despite intensive structural and functional investigations the mechanistic basis of the proficiency of some DNA polymerases to bypass certain lesions and the failure of other DNA polymerase remains elusive. The goal of the applied project is to obtain fundamental new insights into the processes that govern for differential substrate tolerance among DNA polymerases. Our approach is based on switching a DNA polymerase that is deficient of pyrimidine dimer bypass activity (i.e. Thermus aquaticus DNA polymerase) into a lesion bypass-polymerase using iterative high-throughput screening of enzyme variants diversified by mutagenesis. Additionally, a lesion bypass DNA polymerase (i.e. Sulfolobus solfataricus P2 DNA polymerase IV) will be converted into an enzyme that lacks lesion bypass ability albeit retaining high activity on non-damaged DNA. Comparison of the evolved variants with the wild-types by in-depth functional and structural analysis will provide a basis for the understanding of the underlying mechanisms that govern for differential substrate tolerance among these enzymes.

环境胁迫如紫外线损伤DNA，例如通过形成嘧啶二聚体。长期以来，细胞如何通过这些病变进行DNA合成一直不清楚。直到最近，新的专门的DNA聚合酶被确定为参与translesion合成。这些酶存在于多种生物体中，例如参与抑制人类皮肤癌。尽管深入的结构和功能研究，一些DNA聚合酶绕过某些病变的能力和其他DNA聚合酶的失败的机制基础仍然难以捉摸。应用项目的目标是获得基本的新见解的过程中，管理不同的底物之间的DNA聚合酶的耐受性。我们的方法是基于开关的DNA聚合酶，这是缺乏嘧啶二聚体旁路活性（即水生栖热菌DNA聚合酶）到病变旁路聚合酶使用迭代的高通量筛选多样化的诱变酶的变体。此外，病变旁路DNA聚合酶（即硫磺硫化叶菌P2 DNA聚合酶IV）将转化为缺乏病变旁路能力的酶，尽管保留了对未受损DNA的高活性。通过深入的功能和结构分析比较进化的变体与野生型，将为理解这些酶之间差异底物耐受性的潜在机制提供基础。