The mechanism of DNA strand exchange by serine recombinases

All living organisms contain immensely long double-helical DNA molecules that carry the information each cell needs to grow and multiply. This information is encoded in the sequence of the DNA building blocks called bases. Cellular machines translate the code into useful molecules such as proteins. Sometimes a cell changes the set of proteins that it makes in order to acquire new characteristics. For example, a disease-causing bacterium infecting a human being may switch the set of proteins displayed on its surface as a diguise to avoid detection and destruction by the human immune system. One way to do this is to rearrange (or 'edit') the DNA sequence by moving sections from place to place, or by adding/deleting sections. A special class of proteins called site-specific recombinases is dedicated to this 'cutting and pasting' role. To do it properly, the recombinase must identify the precise places in the DNA sequences that must be cut, then cut both DNA strands in each place and rejoin the cut ends in the required new arrangement. One family of site-specific recombinases has been proposed to do this reaction by a remarkable mechanism in which the DNA strands are first cut, then turned on a 'molecular wheel' to bring the cut ends into a new arrangement before rejoining them. This mechanism would be unprecedented in the world of enzymes, and has implications for the design and function of molecular machines, but it has not been proved. It is therefore very important to find out if this is really how the DNA is rearranged. In this project, we will use novel methods where we can see what is happening to the tiny individual recombinase 'machines' that are doing the reaction. We will attach pairs of fluorescent molecules to parts of the DNA in each machine, which we can see with a special type of microscope, and which will flash on and off as the bits of DNA move closer together or farther apart. We will also see what happens when we attach bits of the protein in the machine to the DNA to restrict their movement relative to each other. These experiments will allow us to find out the real mechanism of the reaction and analyse its properties, such as how fast it goes and what can interfere with it.

The serine recombinases make up a large and diverse family of enzymes that bring about DNA site-specific recombination and transposition. Recombination takes place within a protein-DNA complex in which two DNA sequences ('sites') that are to recombine are synapsed by a recombinase tetramer. The mechanism of serine recombinase-mediated recombination has long been controversial. Biochemical and topological studies suggested a 'subunit rotation' mechanism, where the DNA strands are cleaved and then one half of the intermediate protein-DNA complex rotates 180 degrees relative to the other half. Until recently, this mechanism was difficult to reconcile with available structural data, and alternative mechanisms were proposed which would accomplish the same transformation of the DNA but without repositioning of whole subunits. However, a new crystallographic structure of an intermediate in recombination reveals a flat hydrophobic protein interface that may be compatible with subunit rotation. In this project we will carry out experiments designed to distinguish between subunit rotation and other strand exchange models. We will also determine properties of the reaction including the rate of individual steps, stability of intermediates, and sensitivity to modification of the protein or DNA substrate. In one approach we will observe reactions involving recombinase subunits that are fixed to the DNA substrate by site-specific crosslinks or high-affinity DNA-binding domains. A complementary approach will involve the analysis of single reactive protein-DNA complexes using innovative single-molecule fluorescence resonance energy transfer (FRET) methods.