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

环境压力，如紫外线，会损害DNA，例如形成嘧啶二聚体。长期以来，人们一直不清楚细胞是如何通过这些病变进行DNA合成的。直到最近，新的特殊DNA聚合酶才被发现参与翻译合成。这些酶存在于多种生物体中，例如参与抑制人类皮肤癌。尽管进行了深入的结构和功能研究，但一些DNA聚合酶绕过某些病变和其他DNA聚合酶失败的机制基础仍然难以捉摸。应用项目的目标是获得对DNA聚合酶之间差异底物耐受性的过程的基本新见解。我们的方法是基于将缺乏嘧啶二聚体旁路活性的DNA聚合酶（即水热菌DNA聚合酶）转换为病变旁路聚合酶，使用迭代高通量筛选通过诱变多样化的酶变体。此外，病变旁路DNA聚合酶（即solfataricus Sulfolobus P2 DNA聚合酶IV）将被转化为一种缺乏病变旁路能力的酶，尽管对未受损的DNA保持高活性。通过深入的功能和结构分析，将进化的变体与野生型进行比较，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。

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.