Replication repair in real life: analysing how broken DNA replication machines are rebuilt inside cells.

DNA encodes the genetic blueprint that provides the basis for all life. Any corruption of this blueprint can have very harmful consequences for the organism since it leads to mutations that can alter how cells function or even lead to death. In humans such mutations can lead to genetic diseases, cancer and possibly the debilitating effects of old age. Every time a cell divides it must copy its DNA so that each daughter cell receives an accurate, complete set of genetic instructions but unfortunately this copying process also has the potential to corrupt the blueprint. This corruption can occur because the nanomachines that duplicate DNA continually encounter unavoidable obstacles to their movement. Many of these obstacles can be cleared or bypassed, allowing the original nanomachine to continue on its way. However, some of these obstacles do lead to breakdown of the nanomachine. These breakdowns are potentially disastrous since they may lead to parts of the genetic code not being copied or miscopied prior to cell division, resulting in corruption of the code.

These breakdowns, and the risks associated with them, are a problem that all organisms must face. Consequently mechanisms have evolved to reassemble the copying nanomachines back onto the DNA, allowing copying to resume and so reducing the risk of mistakes being introduced into the genetic code. The means by which these replicating nanomachines are reloaded back onto chromosomes appear to be similar in all organisms, a reflection of the conserved nature of the genetic material and the machines that copy this material. However, it is only in bacteria that the enzymes responsible for reloading these nanomachines back onto DNA have been identified. The bacterium Escherichia coli has provided a great deal of information about how this reloading occurs and has provided a model with which to understand how this repair occurs in all organisms. However, in spite of two decades of study, we still know very little about how this repair actually occurs inside cells. This ignorance is in part because of the many different types of obstacle encountered inside cells that may lead to breakdown of these nanomachines and also the complicated, overlapping nature of this nanomachine repair.

To address this gap in our understanding of how the copying of DNA is achieved inside cells, we will use recently-developed microscopy techniques that allow single molecules to be visualised and tracked inside living cells. We will exploit the information available in the bacterium E. coli to monitor nanomachine repair as it happens inside cells. This information will allow us to generate a model of how the rebuilding of copying machines underpins duplication of the genetic blueprint in this simple system, providing insight into how this essential repair occurs inside more complicated organisms such as ourselves.

All organisms strive for high fidelity genome duplication and genetic stability but there are many potential barriers that can halt movement of the DNA replication machinery and threaten completion of genome duplication. Breakdown of a replication fork demands that the replication machinery be reloaded to complete genome duplication. Reloading occurs away from normal replication origins and so specialised reinitiation mechanisms are needed, many features of which are shared between bacteria and eukaryotes. But replisome reloading sometimes requires significant processing of the DNA at the fork prior to reloading, a process that can go awry and lead to genome rearrangements. The balance between replication blockage, accurate replication restart and genetic instability is therefore a fine one.

In spite of the importance of replisome reloading, the enzymes responsible have been identified only in bacteria. Much is known about mechanisms of E. coli PriA- and PriC-catalysed replisome reloading in vitro but we know little about how these two systems underpin genome duplication in vivo: the many different types of replicative barrier inside cells and the overlapping nature of different repair mechanisms make it difficult to deconvolute in vivo ensemble data. We will exploit the information available in E. coli to analyse replication repair one molecule at a time inside cells using sophisticated genetic tools coupled with advanced biophotonics approaches (slimfield and photoactivated localisation microscopy). We will establish how reloading enzymes interface with the replication machinery, determine which types of replicative barrier are addressed by PriA and PriC and probe the links between damaged fork processing and replisome reloading. These analyses will provide a direct view of how replication repair is effected within the harsh environments found within cells and provide a foundation for understanding how such repair occurs in all organisms.

This proposal seeks to understand how the many problems that are encountered by DNA replication machines are overcome to ensure completion of genome duplication. The aim is to provide a mechanistic basis for the ability of cells to survive and divide successfully in the face of potentially lethal breakdowns in DNA replication. The proposal also aims to probe directly the links between replication blockage, blocked fork processing and genome instability. These fundamental studies will provide insight into how cells can overcome replicative stress and go on to divide successfully and, conversely, how genetic instability can result from occasional errors in replication repair.

The fundamental mechanisms of replisome reloading appear to be conserved from bacteria to higher organisms, although it is only in bacteria that mechanistic information on replisome reloading is available. Work over the last 15 years has highlighted the importance of mistakes made during replication repair in the development of genetic disease and other mutation-driven problems such as cancer. Clinicians and scientists with interests in the origins of human and animal genetic diseases will therefore benefit from the fundamental studies proposed here on how replication machines are rebuilt inside cells. Such studies have provided many paradigms for understanding these processes in more complex systems. Examples include the identification in E. coli of replisome reloading away from normal origins, enzyme-catalysed unwinding of damaged replication forks and the importance of nucleoprotein complexes in replisome blockage.

A second set of beneficiaries are those with interests in how to prevent cell division via inhibition of genome duplication. Prevention of replisome reloading in E. coli is lethal, reflecting the frequencies with which replisomes require reloading back onto the chromosome after breakdown. This reloading is also important for the maintenance of cell division in other bacteria and in eukaryotes. Thus the proposed work may be of long-term benefit to pharmaceutical companies and academics with interests in developing new targets for inhibition of disease-causing organisms such as bacteria. Inhibition of replication repair also provides a potential strategy to retard the unregulated cell division that occurs in cancer cells. Our proposed experiments may impact therefore upon the development of novel anti-cancer therapies. Replication repair also contributes to the ability of a subpopulation of bacterial cells to survive exposure to otherwise lethal levels of antibiotics, so-called persistence. Thus our proposed study may inform strategies to undermine persistence mechanisms in bacteria.

This proposal will apply cutting-edge biophotonics to detect single molecules in live cells, requiring the use of bespoke instrumentation that is not currently available commercially. Direct visualisation of complex, partially redundant reaction pathways inside live cells is technically very demanding but critical for the unambiguous dissection of such processes in vivo. Given the growing interest in such imaging techniques, our proposed experiments may inform the development of commercial instrumentation aimed at this type of bioimaging.

The research staff on this project will receive cross-disciplinary training and contribute to the scientifically-literate workforce within the UK. This will benefit the UK economy by enhancing competitiveness in technology-driven industries. Understanding how healthy organisms underpin cell division, and what happens when these processes go awry, will also have long-term benefits to the health and well-being of the UK population. The researchers will also be very well-placed to engage with the public on links between genomes, mutation and the genetic basis of disease, topics of general interest to the public.