Directed evolution of DNA methyltransferases

DNA甲基转移酶（MTases）允许诱变和筛选技术的特殊组合，这将使我们能够筛选多达1012个酶变体，使这种方法成为蛋白质体外定向进化的最强大的应用之一。为了更好地理解蛋白质和酶识别DNA的机制，这是所有生物体中基因调控的基础，我们将尝试以不同的方法改变DNA MTases的DNA识别特异性。此外，我们的研究结果将进一步加深我们对这类迷人的酶的分子机制的了解。在第二组实验中，我们将尝试提高真核MTase结构域的催化效率，目的是了解这些酶的调节原理。DNA的错误甲基化是许多人类癌症的原因。最后，我们将尝试产生MTase变体，其适合于生物技术应用并且特异性地甲基化半甲基化CG位点。这种酶可能在生物医学和生物技术中有很大的用途，因为它将允许开发一种甲基化保留PCR -一种迄今为止根本无法获得的技术。鉴于MTases在定向进化中的特殊优势，我们的研究结果将有助于确定体外进化方法作为重新设计生物分子复杂特性的工具的普遍适用性，并估计这种方法的未来前景。

DNA methyltransferases (MTases) allow special combinations of mutagenesis and screening technologies, which will enable us to screen up to 1012 enzyme variants making this approach one of the most powerful applications of directed in vitro evolution of proteins. In order to better understand the mechanism of DNA recognition by proteins and enzymes, which underlies gene regulation in all organisms, we will try to change the DNA recognition specificity of DNA MTases in different approaches. In addition, our results will further our knowledge on the molecular mechanism of this fascinating class of enzymes. In a second groups of experiments, we will try to improve the catalytic efficiency of eukaryotic MTase domains with the aim to understand the principles of regulation of these enzymes. Erroneous methylation of the DNA is causative for many human cancers. Finally, we will try to generate an MTase variant, that is suitable for biotechnical applications and specifically methylates hemimethylated CG sites. Such enzyme could be of great use in biomedicine and biotechnology, since it would allow to develop a methylation retaining PCR - a technique that so far is not available at all. Given the special advantages of MTases for directed evolution, our results will help to define the general applicability of in vitro evolution methods as a tool to redesign complex properties of biomolecules and to estimate future prospects of this approach.