Recruitment of DNA repair enzymes to stalled transcription complexes

修复受损DNA的机制对所有生命形式的生存至关重要。DNA分子是由四种不同类型的小分子组成的长链，称为核苷酸。这四种类型的核苷酸在DNA上沿着排列的顺序携带着许多不同类型的信息：基因是核苷酸序列，含有制造特定类型蛋白质所必需的信息;其他一些核苷酸序列是“开关”，控制特定基因何时何地开启;其他的序列给出了复制新的DNA分子的指令，等等。每个细胞中的DNA都在不断地受到攻击，来自阳光中的紫外线辐射，来自环境中的诱变化学物质，来自电离辐射，甚至是细胞自身代谢的副产品。DNA中核苷酸的化学修饰可以改变或破坏它们携带的信息，如果没有修复DNA受损部分的机制，细胞基因组中的信息将迅速被破坏，以至于细胞无法发挥功能。各种DNA修复系统已经进化来抵消DNA的不断破坏。其中大多数使用酶的组合来结合受损的核苷酸，切除DNA的受损部分，并用新核苷酸的“补丁”取代它。用于修复受损DNA的系统取决于已发生的损伤类型和发生损伤的DNA类型。当开启的基因中发生DNA损伤时，修复酶面临着特殊的困难。为了使基因中的信息被细胞利用，该基因的信使RNA拷贝必须由称为RNA聚合酶的酶在称为转录的过程中产生。RNA聚合酶将双螺旋DNA分子的两条链分开，并使用一条链作为合成信使RNA的模板。如果被复制的链上的核苷酸被损坏，RNA聚合酶就会被卡住。在损伤被修复之前，基因不能转录，但是由于DNA损伤被RNA聚合酶固定，DNA修复酶无法到达它来修复它，这对细胞来说是潜在的致命性。被称为转录修复偶联因子的专门蛋白质克服了这个问题，因此，阻断活性基因中RNA聚合酶的DNA损伤比DNA其他区域的DNA损伤更快地修复。这项研究的目的是了解转录修复偶联因子如何加快活性基因中DNA修复的速度。该项目利用来自细菌模型系统的RNA聚合酶和修复酶。我们之前已经研究了这种转录修复偶联因子如何从受损的DNA中去除堵塞的RNA聚合酶。在这项工作中，我们将研究转录修复偶联因子和DNA修复蛋白之间的相互作用如何导致损伤被修复更快，当它遇到RNA聚合酶比它是当修复蛋白自己的功能。在这个模型系统中学到的经验教训将有助于我们理解类似蛋白质在更复杂的生物体中发挥作用的方式。操纵DNA的蛋白质在许多基本的细胞过程中起着关键作用，在生物技术中也有重要的应用。通过深入了解这些蛋白质的功能方式，我们的目标是有助于增加对疾病的理解和设计新的治疗策略。

Mechanisms for repairing damaged DNA are essential for the survival of all forms of life. DNA molecules are long strands made up of four different types of small molecule, called nucleotides. The order in which these four types of nucleotide are arranged along the DNA carries many different types of information: genes are sequences of nucleotides that contain the information necessary for making a particular types of protein; some other sequences of nucleotides are 'switches' that control when and where particular genes are turned on; other sequences give instructions about making a new copy of the DNA molecule, and so on. The DNA in every cell is under constant attack, from the ultraviolet radiation in sunlight, from mutagenic chemicals in the environment, from ionising radiation, and even from the by-products of the cell's own metabolism. Chemical modification of the nucleotides within DNA can change or destroy the information that they carry, and without mechanisms for repairing damaged sections of DNA the information within a cell's genome would rapidly become so corrupted that the cell would be unable to function. A variety of DNA repair systems have evolved to counteract the constant damaging of DNA. Most of these use a combination of enzymes to bind to the damaged nucleotide(s), cut out the damaged section of DNA and replace it with a 'patch' of new nucleotides. The system that is used to repair damaged DNA depends on both the type of damage that has occurred, and the type of DNA that it occurs in. The repair enzymes face particular difficulties when DNA damage occurs in a gene that is turned on. In order for the information in a gene to be used by the cell a messenger RNA copy of the gene must be made by an enzyme called RNA polymerase, in a process called transcription. RNA polymerase separates the two strands of the double-helical DNA molecule, and uses one strand as a template for the synthesis of messenger RNA. If nucleotides on the strand being copied have been damaged, RNA polymerase can become jammed. The gene cannot be transcribed until the damage is repaired, but because the DNA damage is lodged within RNA polymerase the DNA repair enzymes cannot reach it to repair it. This is potentially lethal for the cell. Specialised proteins called transcription-repair coupling factors overcome this problem, and as a result DNA damage that blocks RNA polymerase in active genes is repaired more quickly than DNA damage in other regions of DNA. The purpose of this research is to understand how a transcription-repair coupling factor speeds up the rate of DNA repair in active genes. This project utilises RNA polymerase and repair enzymes from a bacterial model system. We have previously investigated how this transcription-repair coupling factor removes the jammed RNA polymerase from the damaged DNA. In this work we will investigate how the interactions between the transcription-repair coupling factor and the DNA repair proteins lead to damage being repaired more quickly when it is encountered by RNA polymerase than it is when the repair proteins function on their own. The lessons learnt in this model system will contribute to our understanding of the way that similar proteins may function in more complex organisms. Proteins that manipulate DNA play critical roles in many fundamental cellular processes and also have important applications in biotechnology. By gaining a thorough understanding of the ways in which such proteins function we aim to contribute to increased understanding of disease and the design of novel therapeutic strategies.