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

点击翻译按钮获取中文摘要

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.