Application of single-enzyme kinetics to investigate the turnover rate, processivity and specificity of DNA methyltransferase 1

点击翻译按钮获取中文摘要

DNMT1 is the main enzyme responsible to copy DNA methylation patterns in human cells, which have fundamental roles in development and disease. It is a large multidomain Protein which is regulated by conformational changes and at least two auto-inhibitory domains. We will employ novel approaches, single enzyme kinetics with long hemimethylated DNA substrates and conformationally locked DNMT1 mutants, to solve fundamental mechanistic questions of DNMT1 and relate its biochemical properties with its biological function in the cell. Key questions of this application are to determine the catalytic rate of DNMT1 during processive DNA methylation, the specificity of DNMT1 for hemimethylated CpG sites, the role of interacting proteins and post translational modifications in the regulation of activity and specificity and the influence of conformational changes in these processes. Our preliminary data indicate that this project will provide important new insights into the function and mechanism of this essential DNA modification enzyme. Our project addresses major research questions in the field of molecular epigenetics namely the detailed molecular mechanism of the accurate inheritance of DNA methylation patterns and the process of epigenetic information transfer in general. Moreover, the project addresses a major challenge in molecular enzymology, which is the question how the activity of large multidomain enzymes are allosterically regulated by conformational changes and binding of interacting proteins.