Award details

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

Reference	BB/H001212/1
Principal Investigator / Supervisor	Professor Margaret Smith
Co-Investigators / Co-Supervisors	Professor James Naismith
Institution	University of Aberdeen
Department	School of Medical Sciences
Funding type	Research
Value (£)	413,066
Status	Completed
Type	Research Grant
Start date	01/09/2009
End date	31/12/2012
Duration	40 months

Abstract

The integrase encoded by the Streptomyces temperate phage, phiC31, mediates both integration and excision. In the absence of accessory factors, phiC31 integrase is unidirectional i.e. it only recombines attP x attB in an integration reaction to form the products, attL and attR. As phiC31 integrase is a serine recombinase it has the same (remarkable) mechanism of DNA cleavage and strand exchange as other serine recombinases. Our work is aimed at understanding the control of integrase activity, which we propose involves subtle differences in protein conformation as a result of protein-DNA interactions with different recombination sites. Integrase has a large C-terminal domain (CTD; ~450 aa) that is required for DNA binding, synapsis of attP and attB and prevents integrase from performing the excision reaction in the absence of accessory factors. Native integrase can only synapse attP and attB. We propose that integrase adopts different conformations on attP and attB that enable synapsis between these sites only and prevents synapsis between other pairs of sites. The problem is therefore how does integrase recognise its different recombination sites and how does this recognition translate to the synaptic interface? We have isolated mutants that have lost the ability to prevent excision, i.e. bidirectional or hyperactive mutants. These map to a motif in the CTD that appears to have a coiled coil structure. Preliminary data suggests that this coiled coil motif is central to the recognition of attP and attB and transmitting this information to a synaptic interface. The objectives are to test the predictions on the structure of the putative coiled coil motif and how it interacts with other integrase functions. Information will also be sought on how integrase binds to and discriminates between attP and attB.

Summary

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 bringsthe 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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	Synthetic Biology
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search