SANDPIT : Knots and Evolution - Topologically Driven Integrase Mutagenesis

自从20世纪60年代末发现DNA结和链以来，人们发现DNA结和链在许多细胞过程中发挥着关键作用。因为它们无处不在，所有的有机体都开发出了特殊的蛋白质，其功能是帮助解开DNA的结和链接。还有其他重要的蛋白质--称为重组酶--可以改变DNA碱基序列的顺序。虽然重组酶的主要功能是重新排列碱基对的顺序，但在此过程中，它们往往会引起DNA打结或连接的变化。由于所有这些原因，分子生物学家对研究结和环产生了兴趣。自19世纪末以来，数学家们出于自己的原因一直在研究结，与DNA无关。从数学上讲，结是一个位于三维空间中的一维物体，就像标准的圆一样，但我们不能将其平滑地变形为标准的圆。纽结和链环的数学理论被证明是非常丰富和令人惊讶的复杂的，并且与一般的三维空间密切相关，称为三维流形。(对这些空间的研究称为3-流形拓扑。)尽管这个主题非常深刻，但一些最简单的问题仍然没有答案：即使在今天，如果你给世界顶尖的结理论家两个足够复杂的结，他们也没有已知的算法可以总是用来判断一个结是否可以变形成另一个。利用纽结理论中的工具，数学家已经能够帮助生物学家更好地了解一些蛋白质与DNA相互作用的方式。例如，数学家已经建立了重组酶蛋白质如何重新洗牌DNA序列的模型。(1)然后，这些模型可以预测这些相互作用的各种新特征--例如，当蛋白质连接时DNA采取的特殊几何构型，或者反应通过什么生化途径进行。位点特异性重组酶介导DNA序列的重新洗牌非常重要，因为它在广泛的生物学过程中起着关键作用，并且是细菌进化的重要机制，例如最近出现的整合子介导的多重抗生素耐药性。整合子整合酶的不同寻常之处在于它们进行了广泛的重组反应，预计将产生各种不同的拓扑结构不同的产物。打结产物的形式将指示已发生的复合反应的类型和频率。为了深入了解整合酶的进化、整合子驱动的基因组可塑性和细菌进化的基本机制，将对一些不同的系统发育和进化的不同整合酶进行机械研究，并确定其产物的拓扑结构。DNA可以形成非常复杂的结。但在所有可能的非常复杂的结中，只有一小部分以DNA结的形式出现。其中一个问题是(2)确定重组酶作用于DNA结构型的初始家族后，哪些结会出现。在这项提案中，我们将探索这两个领域(1)和(2)的一个大的和重要的蛋白质家族，整合酶。为了回答这些问题，我们将使用来自3-流形拓扑的尖端技术，并结合新颖的微生物实验。答案将帮助我们更全面地理解这些重要的进化动因。

Since their discovery in the late 1960s, DNA knots and links have been found to play key roles in hosts of cellular processes. Because they are so ubiquitous all organisms have developed special proteins whose function is to help untie DNA knots and links. There are also other important proteins-- called recombinases -- that can alter the order of the sequence of the DNA basepairs. While the main function of recombinases is to rearrange the order of basepairs, in the process of doing this they often cause changes to DNA knotting or linking. For all these reasons molecular biologists became interested in learning about knots and links. Mathematicians have studied knots since the late 19th century for their own reasons, having nothing to do with DNA. Mathematically a knot is a one-dimensional object sitting inside 3-space, just like a standard circle does, but which we cannot smoothly deform to a standard circle. The mathematical theory of knots and links turns out to be very rich and surprisingly complicated, and intimately related to general 3-dimensional spaces, called 3-manifolds. (The study of these spaces is called 3-manifold topology.) Although the subject is very deep, some of the simplest questions remain unanswered: even today if you hand the world's top knot theorists two sufficiently complicated knots there is no known algorithm they can use to always tell whether one knot can be deformed into the other. Using tools from knot theory, mathematicians have been able to help biologists better understand the ways some proteins interact with DNA. For example, mathematicians have developed models of how the recombinase proteins reshuffle the DNA sequence. (1) These models can then predict various new features of these interactions - e.g. particular geometric configuration the DNA takes when the protein is attached or what biochemical pathway the reactions proceeds through. Site-specific recombinases mediate the reshuffling of the DNA sequence is important because of its key role in a wide variety of biological processes and is an important mechanism for bacterial evolution e.g. the recent emergence of multiple antibiotic resistance mediated by integrons. The integron integrases are unusual in that they undertake a wide variety of recombination reactions and it is anticipated that there will be a wide variety of topologically distinct products generated. The form of the knotted products will be indicative of the type and frequency of recombination reactions that have occurred. A number of phylogenetically and evolutionary distinct integrases will be mechanistically studied and the topology of their products determined in order to gain insight into integrase evolution, the fundamental mechanisms of integron driven genome plasticity and bacterial evolution. DNA can form very complicated knots. But only a small fraction of all possible very complicated knots appear as DNA knots. One issue has been (2) determining which knots can show up after a recombinase acts on an initial family of DNA knot configurations. In this proposal we will explore these two arenas (1) and (2) for a large and important family of proteins, the integrases. To answer these questions, we will use cutting-edge techniques from 3-manifold topology, combined with novel microbiological experiments. The answers will help us understand these important evolutionary agents more completely.