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

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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.