Replication fork repair at the single-molecule level

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

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.

Technical Summary

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.

Publications

10 25 50
 
Description Forked DNA structures are key intermediates in DNA replication. 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 project aimed to test the hypothesis that binding of replication repair enzymes to branched DNA intermediates shifts the equilibrium between different conformations. We also aimed to test the hypothesis that PriA helicase, the key replication fork reloading enzyme in E. coli, functions at forks by translocating along the lagging strand template whilst remaining bound to the branch point of the fork, generating a loop of ssDNA for subsequent replicative helicase loading. This proposal takes advantage of multi-parameter fluorescence detection (MFD) to study these DNA-protein interactions at the single-molecule level.

Central to all replication fork repair is the binding of repair enzymes to forked DNA structures. Interaction of proteins with their DNA substrates is governed in part by the population of conformations adopted by the DNA prior to protein binding but little is known of the conformations adopted by forked DNA structures. We demonstrated that the combination of MFD of single molecules and molecular dynamics (MD) simulations could reveal the high-resolution global structure of a branched DNA molecule in solution, free from heterogeneity and surface effects [1]. The MFD system consists of a confocal microscope with pulsed laser excitation and simultaneous detection in four channels (two-colour for both parallel and perpendicular polarisation) using sensitive photon-counting detection. We studied a branched DNA molecule, which we term a four-stranded fork (4SF), designed to mimic the possible structure of a blocked replication fork. By labelling with dyes at multiple positions and performing single-molecule Förster resonance energy transfer (SM-FRET) measurements, we were able to determine multiple distance constraints for modelling. We found that it adopts an open, planar conformation, without coaxial stacking of arms and with evidence for ion-induced folding via interactions with Mg2+ ions.

We extended this approach to two- and three-stranded forks (2SF and 3SF), which are constructs that have ssDNA at the branchpoint. For the 2SF, which has two single-strands at the branch, we observed inter-strand interactions, but only under low-salt conditions, which we attributed to transient formation of base pairs (manuscript in preparation). The MFD data indicated that these interactions are very heterogeneous. The above structural data provided a baseline for our DNA-protein studies. We then analysed interactions of forked DNA (3SF) with the PriA helicase. We first demonstrated binding using the MFD approach; we detected changes in the anisotropy of the donor dye upon PriA binding. We then showed that addition of ATP to a PriA-DNA complex results in the rapid formation of a high FRET state, which we have attributed to the looping mechanism discussed above (manuscript in preparation). We also studied the related three-way DNA junction [2]. In this case, we made the surprising finding that the branchpoint was not fully base paired, in spite of the full Watson-Crick complementarity. This has implications for the interactions of proteins with such junctions. In addition, such junctions are widely used as nanostructural components and as supramolecular components. Therefore, we anticipate that this work will have an impact far beyond the field of DNA-protein interactions.

[1] Sabir, T., Schröder, G.F., Toulmin, A., McGlynn, P. & Magennis, S.W. (2011) J. Am. Chem. Soc. 133, 1188-1191. [2] Sabir, T., Toulmin, A., Ma. L., Jones A.C., McGlynn, P., Schröder, G.F. and Magennis, S.W. J. Am. Chem. Soc. (2012) 134, 6280-6285.
Exploitation Route We have provided important information on the structure of branched DNA structures, which has relevance to medicine and nano science.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Dr. Gunnar Schröder 
Organisation Julich Research Centre
Country Germany 
Sector Public 
PI Contribution We performed single-molecule FRET measurements to produce intramolecular distance restraints for modelling of branched DNA.
Collaborator Contribution Dr. Schröder performed MD simulations of branched DNA using the FRET distance restraints provided by us.
Impact 10.1021/ja211802z
Start Year 2010
 
Description Prof. Anita Jones 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed single-molecule fluorescence measurements of branched DNA molecules.
Collaborator Contribution They performed time-resolved ensemble fluorescence measurements of branched DNA molecules
Impact 10.1021/ja211802z