Bacterial DNA Methyltransferases

9983125改变DNA结构的酶执行生命所必需的功能。DNA甲基转移酶通过修饰特定的腺嘌呤和胞嘧啶来扩大DNA的信息含量。DNA甲基化是基因表达的表观遗传控制的最常见形式，发生在从细菌到人类的生物体中。这是一种重要的修饰，其机制尚未完全了解。 本项目使用细菌酶来解决这个问题，试图了解序列特异性DNA修饰的分子基础。DNA甲基化酶在催化之前使DNA弯曲并翻转出它们的靶碱基。该项目建立在由NSF资助并在本PI实验室进行的最新发现的基础上，该发现允许真实的跟踪DNA弯曲和碱基翻转。这一研究领域，DNA修饰酶的构象机制的研究，只有通过使用PI开创的几种动力学和光谱方法并与合作者一起才是可行的。如果我们要理解这些酶如何区分DNA序列，并执行其特定的化学反应，那么了解这种构象变化的潜在机制至关重要。最重要的是，其他方法，如X射线晶体学，不能提供必要的定量和功能的见解，因为它们不能提供动态信息。这一信息对于理解构象转变到酶特异性是非常重要的。 这项研究提供的见解应该对理解其他酶的作用机制有影响，例如参与DNA修复的酶，因为它们中的许多酶似乎也使用类似的构象机制，正如其他研究人员使用类似于这里开创的方法所揭示的那样。

9983125ReichEnzymes that alter the structure of DNA perform functions that are essential for life. DNA methyltransferases expand the information content of DNA by modifying specific adenines and cytosines. DNA methylation is the most common form of epigenetic control of gene expression and occurs in organisms ranging from bacteria to man. This is an essential modification whose mechanism is not completely understood. This project addresses this topic using bacterial enzymes in an attempt to understand the molecular basis of sequence-specific DNA modification. DNA methylases bend the DNA and flip out their target bases prior to catalysis. This project builds on recent discoveries funded by NSF and conducted in this PI's laboratory, that allow the tracking of both DNA bending and base flipping in real time. This research area, the study of conformational mechanisms of DNA modifying enzymes, is only feasible through the use of several kinetic and spectroscopic methods pioneered by the PI and in conjunction with collaborators. Understanding the underlying mechanisms of such conformational changes is critical if we are to appreciate how such enzymes discriminate among DNA sequences, and carry out their particular chemistries. Most importantly, other methods such as x-ray crystallography do not provide the necessary quantitative and functional insights since they cannot provide dynamic information. This information is clearly important to gain an understanding of conformational transitions to enzyme specificity. The insights provided by this research should have implications for understanding the mechanism of action of other enzymes such as those involved in DNA repair since many of them also appear to use similar conformational mechanisms, as revealed by other investigators using methods similar to those pioneered here.