Recruitment of DNA repair enzymes to stalled transcription complexes

修复受损DNA的机制对于所有生命形式的生存都是必不可少的。DNA分子是由四种不同类型的小分子（称为核苷酸）组成的长链。这四种核苷酸沿着DNA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，包含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控制特定基因何时何地开启的“开关”；其他的序列给出了制造DNA分子新拷贝的指令，等等。每个细胞中的DNA都不断受到攻击，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，甚至来自细胞自身代谢的副产品。DNA内核苷酸的化学修饰可以改变或破坏它们所携带的信息，如果没有修复DNA受损部分的机制，细胞基因组内的信息将迅速被破坏，细胞将无法发挥功能。各种各样的DNA修复系统已经进化出来，以抵消DNA的持续破坏。其中大多数使用一种酶的组合来与受损的核苷酸结合，切掉受损的DNA部分，并用新核苷酸的“补丁”取而代之。用于修复受损DNA的系统取决于已经发生的损伤类型和发生损伤的DNA类型。当DNA损伤发生在一个开启的基因中时，修复酶面临着特别的困难。为了使基因中的信息被细胞利用，基因的信使RNA副本必须由一种叫做RNA聚合酶的酶合成，这个过程叫做转录。RNA聚合酶分离双螺旋DNA分子的两条链，并使用其中一条链作为合成信使RNA的模板。如果被复制链上的核苷酸被破坏，RNA聚合酶就会被阻塞。在损伤被修复之前，基因不能被转录，但由于DNA损伤位于RNA聚合酶内，DNA修复酶无法到达修复它。这对细胞来说可能是致命的。被称为转录修复偶联因子的特殊蛋白质克服了这一问题，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损伤修复得更快。本研究的目的是了解转录修复偶联因子如何加速活性基因中的DNA修复速率。本项目利用细菌模型系统中的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RNA聚合酶遇到损伤时比修复蛋白自身功能时更快地修复损伤。从这个模型系统中吸取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可能起作用的方式。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，我们的目标是促进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.