Replication fork repair at the single-molecule level

The ability of cells to duplicate millions of base pairs of DNA every time they divide, DNA replication, is one of the wonders of biology. Equally remarkable is the accuracy with which this complex process is achieved, with less than one mistake made for every million bases of DNA copied. This feat is achieved through the subtle interplay of the DNA with enzymes, which are protein molecules that act as the workhorses of the cell, catalyzing all cellular processes. Many proteins are involved in DNA replication and they work together forming the replication machinery of the cell. Any mistakes (mutations) made during replication can be corrected by enzymes that inspect the DNA and make alterations if necessary (just as machines check for defects on a factory production line). In addition to these kinds of mistakes, the machinery can also encounter blocks on the DNA (analogous to a jammed zip fastener). These can take the form of chemical damage to the DNA caused by products generated internally during metabolism (i.e. byproducts from certain foods) or by a wide range of outside agents such as tobacco smoke or the ultraviolet component of sunlight. Without specialised enzymes such mutations would not be recognized and repaired, which could lead to the death of the cell or, in higher organisms such as man, the onset of cancer. To understand the mechanisms that underpin DNA replication, we are utilising advanced techniques to determine how cells try and overcome such blocks. The techniques are based on the phenomenon of fluorescence, which is the emission of light following irradiation and absorption of light of a different colour. The use of fluorescent labels, small molecules that tag DNA, allow the DNA to be visible when light is shone on a sample. Conventional fluorescence measurements, so called ensemble methods, measure a great many molecules simultaneously, and the resultant fluorescence signal is the average of the signal from all labelled DNA molecules. Since complex molecules like DNA and proteins will be doing different things at different times, it is impossible to study complex dynamic behaviour or to separate the signals from different molecules using ensemble techniques. In this project, we will use advanced fluorescence technology that allows molecules to be studied at the single-molecule level, providing unprecedented information about how cells insure against the inevitable breakdowns that are thought to occur in all organisms, including ourselves.

The complex multi-subunit machines that duplicate chromosomes within cells possess high fidelity, high processivity and high speed, properties that ensure faithful passage of a cell's genetic material to its offspring. However, we now know that replication forks encounter a variety of potential barriers to their progression along chromosomes, such as DNA damage and template-bound proteins. Thus it is thought that all organisms must possess replication fork repair systems, though little is known about them at the molecular level. This programme is focused on the study of forked DNA structures at the single-molecule level using advanced fluorescence spectroscopy and aims to take advantage of the full eight-dimensional fluorescence information: intensity, emission spectrum, excitation spectrum, distance between fluorophores (FRET), time, fluorescence quantum yield, fluorescence lifetime, and the polarisation of the emitted light. This approach is known as multi-parameter fluorescence detection (MFD). Studying forked DNA structures at the single-molecule level, and thereby avoiding ensemble averaging, is an extremely powerful approach because these systems are heterogeneous, cannot be synchronised, and may have transient intermediates or undergo rare events. By detecting individual replication machines, the distribution and dynamics of molecular properties and interactions can be determined. In this project we will analyse the conformations adopted by forked DNA structures and test the hypothesis that binding of replication repair enzymes to branched DNA intermediates shifts the equilibrium between different conformations, in effect remodelling DNA forks. We will also interrogate the mechanism of action of one of these enzymes, PriA helicase, by testing the hypothesis that this branched DNA-specific helicase functions at forks by translocating along the lagging strand template whilst remaining bound to the branch point of the fork.