The mechanism of phiC31 integrase; a unidirectional recombinase for genome engineering

要治愈有遗传病的人真的很难。这些疾病是由有缺陷的基因引起的。最好的治疗方法是给他们一种有效的基因。理想情况下，这种治疗只需要在他们的一生中进行一次，因为基因，当它们是染色体的一部分时，会忠实地从一个细胞传递到另一个细胞，因此治疗将会持续下去。虽然这听起来很简单，但实际上很难。这个项目涉及一种将正确基因植入病人染色体的可能方法。一些感染细菌的病毒（称为噬菌体或噬菌体）有办法将自己的基因植入细菌宿主的染色体中。这个过程涉及到一种叫做整合酶的蛋白质，因为它们能将两段DNA整合成一段。大多数整合酶优先使用细菌染色体上的一个特定位点，即atb位点，噬菌体DNA进入该位点。在噬菌体DNA中也有一个优先的位点，attP位点。为了将正确的基因引入人体以治疗疾病，我们需要设计整合酶，使其能够找到一个安全合适的位置，将DNA整合到那个人的染色体中。因此，这个项目是关于理解这些集成是如何工作的，以便我们能够以合理的方式改变它们。例如，我们想知道整合酶是如何被控制的。整合酶的一个特点是，在没有其他噬菌体蛋白的情况下，它是不可逆的。这意味着一旦正确的基因被插入，它就会永远存在，因此只需要一次治疗。事实上，我们所说的不可逆是指它只能在积分反应中使用attP和attB位点。在这个过程中，attP的两个部分被分离并与attB的两个部分结合形成混合位点attL和attR。在这四个位点中，整合酶只与attP和attB发生反应。整合酶在反应途径的早期检测到attP和attB的存在，这一阶段将两个位点结合在一起。结合在两个位点attP和attB上的整合酶之间存在一种锁与键的相互作用，激活通路的其余部分来完成整合。如果没有正确的锁和键相互作用，例如当整合酶结合到两个attP位点，一个attP和一个attL位点或attL和attR位点时，该途径被阻断。我们最近发现了整合酶的一小部分，一个“控制模块”，它是一种机制的一部分，可以感知它所绑定的位点类型，并将这些信息传递给生成“锁”或“钥匙”类型结构。我们发现，截断的整合酶（c端结构域或CTD）缺乏化学反应所需的整合酶部分，但仍包含控制模块和DNA结合活性，可以自行进行锁和钥匙反应。我们已经将整合酶进一步分解为我们所展示的可以绑定到自身的控制模块。这是发生在全长整合酶中的锁与钥匙的相互作用吗？我们将使用更多的突变体来测试关于控制模块如何与自身相互作用的想法，以及这是锁和钥匙的相互作用，还是它是区分DNA结合上attP和attB的传感机制的一部分。我们还将尝试确定集成酶中直接识别attP和attB位点的部分，以及它如何与控制模块相互作用。最后，我们将着眼于整合反应的最开始，即DNA结合的过程，并使用化学和热力学来研究整合酶在attP和attB位点上的足迹是如何不同的。

It is really difficult to cure people with genetic diseases. These diseases are caused by defective genes. The best cure would be to give them a gene that works. Ideally this treatment would need to be given only once in their lifetime because genes, when they are part of the chromosome, are passed faithfully from one cell to the next and so the cure would perpetuate. Although this sounds simple, in practice its very hard. This project concerns a possible way of getting the right gene into a sick person's chromosome. Some viruses that infect bacteria (called bacteriophages or phages) have a way of getting their own genes into the chromosome of their bacterial hosts. This process involves proteins called integrases, because they integrate two pieces of DNA into one. Most integrases use a particular site, an attB site, in the bacterial chromosome preferentially over all others and the phage DNA goes into that site. There is also a preferred site in the phage DNA, the attP site. In order to introduce correct genes into people as a cure for disease, we need to engineer the integrase so that it can find a safe and suitable site for integrating DNA in that person's chromosome. Consequently this project is about understanding how these integrases work so that we can alter them in a rational way. We would like to know, for instance, how integrase is controlled. A feature of integrase is that it is irreversible in the absence of any other phage proteins. This means that once the correct gene is inserted it is there forever, hence the need for only one treatment. In fact what we mean by irreversible is that it can only use the attP and attB sites in the integration reaction. During this process the two halves of attP get split and join up with the two halves of attB site to form hybrid sites attL and attR. Of these four sites integrase only reacts with attP and attB. Integrase detects the presence of attP and attB very early on in the reaction pathway, a stage that brings the two sites together. There is a kind of lock and key interaction between integrases bound to the two sites attP and attB that activates the rest of the pathway to complete integration. Without the right lock and key interaction, such as when integrase is bound to two attP sites, an attP and an attL site or the attL and attR sites, the pathway is blocked. We have recently discovered a small part of integrase, a 'control module', that is part of a mechanism that senses which type of site it is bound to and communicates this information to generate a 'lock' or a 'key' type structure. We have discovered that a truncated integrase (the C-terminal domain or CTD) that lacks the part of integrase necessary for the chemistry of the reaction, but still contains the control module and DNA binding activity, can do the lock and key reaction on its own. We have broken down integrase still further to just the control module which we showed can bind to itself. Is this the lock and key interaction that occurs in full length integrase? We will use more mutants to test ideas about how the control module interacts with itself and whether this is the lock and key interaction or whether it is part of a sensing mechanism that discriminates between attP and attB on DNA binding. We will also try to identify the part of integrase that directly recognises the attP and attB sites and how this interacts with the control module. Finally we will look at the very beginning of the integration reaction, i.e. the process of DNA binding, and use chemicals and thermodynamics to look at how the footprints made by integrase on the attP and attB sites are different.