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

要治愈遗传病患者真的很难。这些疾病是由基因缺陷引起的。最好的治疗方法是给他们一种有效的基因。理想情况下，这种治疗在它们的一生中只需要进行一次，因为当基因成为染色体的一部分时，它们会从一个细胞忠实地传递到下一个细胞，因此治愈将永久化。虽然这听起来很简单，但实际上却很难。这个项目涉及一种可能的方法，将正确的基因植入病人的染色体中。一些感染细菌的病毒(称为噬菌体或噬菌体)有一种方法将自己的基因植入细菌宿主的染色体中。这个过程涉及被称为整合酶的蛋白质，因为它们将两段DNA整合为一段。大多数整合酶优先使用细菌染色体中的一个特定位点，即attB位点，然后噬菌体DNA进入该位点。在噬菌体DNA中也有一个首选的位点，attP位点。为了将正确的基因引入人们体内作为治疗疾病的方法，我们需要对整合酶进行改造，以便它能够找到一个安全和合适的位置来将DNA整合到人的染色体中。因此，这个项目是关于了解这些整合酶是如何工作的，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。整合酶的一个特征是在没有任何其他噬菌体蛋白的情况下是不可逆的。这意味着，一旦正确的基因被注入，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在整合反应中只能使用attP和attB位点。在这个过程中，attP的两半分裂，并与attB位点的两半结合，形成杂交位点attL和attR。在这四个位点中，整合酶只与attP和attB反应。整合酶在反应途径的早期就检测到attP和attB的存在，这一阶段将这两个位点结合在一起。在与attP和attB两个位点结合的整合酶之间存在一种锁和键的相互作用，激活其余的途径以完成整合。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，该途径就会被阻断。我们最近发现了整合酶的一小部分，即一种控制模块，它是一种机制的一部分，它可以感知它结合到哪种类型的站点，并传递这种信息来产生一种‘锁’或‘钥匙’类型的结构。我们已经发现，一个截短的整合酶(C-末端结构域或CTD)，如果缺乏反应化学所需的整合酶部分，但仍然包含控制模块和DNA结合活性，就可以独立完成锁定和关键反应。我们已经进一步分解了整合酶，只有我们展示的控制模块可以绑定到它自己。这是在全长整合中发生的锁和键的相互作用吗？我们将使用更多的突变体来测试控制模块如何与自身相互作用，以及这是否是锁和键的相互作用，或者它是否是在DNA结合上区分attP和attB的传感机制的一部分。我们还将尝试确定整合酶直接识别attP和attB位点的部分，以及这部分如何与控制模块相互作用。最后，我们将看看整合反应的最开始，即DNA结合的过程，并使用化学和热力学来观察整合酶在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.