Why does transcription present a major barrier to genome duplication?

When a cell divides, all of its DNA must be copied so that each of the two new cells contains a complete set of genes. The copying and passing on of genetic information is a central feature of life, and is performed in a similar manner by organisms from bacteria to humans. DNA replication must be accurate because mistakes can corrupt the genetic message with potentially fatal consequences. The DNA replication machinery must also be able to overcome any obstacles that it encounters, because a cell cannot divide until its genome has been fully duplicated. Potential obstacles to DNA replication are common, because DNA within cells is coated with proteins that package, repair or read the genetic material. In this project we aim to determine how the DNA replication machinery deals with a common and potent obstacle: collisions with the molecular machinery that 'reads' the information contained within genes. The central components of this machinery are enzymes called RNA polymerases. These enzymes unwind the double-stranded DNA molecule at the beginning of a gene so that they can use one of the two strands as a template for the construction of a temporary copy, called mRNA. In order to copy a complete gene the RNA polymerase must move along the DNA, unwinding it as it goes. During this process RNA polymerase binds tightly to the DNA to stop it falling off before it reaches the end. The copying of genes by RNA polymerase is regulated: the cell needs more copies of some genes than others, and so RNA polymerases are found rarely on some genes and in nose-to-tail traffic jams on others. Like RNA polymerase, the enzymes that replicate DNA move along, and unwind, the DNA double helix. As the DNA replication machinery has to copy the entire genome it will collide frequently with RNA polymerases that are copying individual genes. Because RNA polymerases bind tightly to DNA, the DNA replication machinery often has difficulty moving past them, and in some cases needs help from other proteins in order to overcome the obstacle. In our preliminary experiments we have identified two proteins that help the DNA replication machinery to push its way through obstacles. We have also shown that one of the main reasons that these proteins are needed by cells is to help overcome the 'roadblock' effect caused by RNA polymerases. The experiments that we now wish to undertake will enable us to discover what determines whether a particular RNA polymerase blocks DNA replication or is easily bypassed (for example, does the length of an RNA polymerase 'traffic jam' determine how easily the replication machinery can get through?). They will also enable us to define the various helper-systems that the cell uses to overcome such obstacles, and understand which systems help at which types of obstacle. There are many reasons why it is interesting and important to undertake this study. These experiments are exciting because they aim to understand the interface between two of the most fundamental and important processes in the cell: the copying and expression of the genetic material. These processes are well conserved in all organisms, and our findings will have implications beyond the experimentally tractable model system in which we will work. The understanding that we will gain will also have practical implications. It is now possible for scientists to construct organisms in which large sections of the genome, or even the entire genome, are artificially designed and constructed. A sound understanding of the interplay between genome replication and gene expression will be important if these designed organisms are to function correctly. Furthermore, by understanding how cells help the DNA replication machinery to overcome obstacles we can identify opportunities to disrupt those processes: drugs that target the helper-systems and prevent complete DNA replication may have applications in anti-bacterial or anti-tumour chemotherapy.

Genome duplication requires high processivity and high fidelity but proteins bound to the DNA template, especially those associated with transcription, present barriers to replication and therefore challenges to genome stability. However, why transcription presents such a problem for replication is unknown. Head-on collisions between transcribing RNA polymerases (RNAPs) and replication complexes are inhibitory but emerging evidence suggests that codirectional collisions are as important in vivo. This may be because arrested RNAPs, formed either by pausing and backtracking or by lesions within the template, may be as important as actively transcribing RNAPs, possibly because arrested RNAPs result in accumulation of RNAP arrays in highly transcribed regions. We aim to establish the features of transcription that present barriers to genome duplication in vivo, to determine the impact of different features of transcription on replisome movement in vitro and to correlate in vitro blockage of replisomes with in vivo impacts on replication fork movement. We will also analyse systematically the mechanisms needed to minimise the impact of transcription on genome duplication. We will correlate the relative importance of transcription/replication elongation factors with the critical features of transcription that pose barriers to replisomes. We have also discovered recently that a helicase known to promote genome duplication in E. coli (UvrD) interacts specifically with RNAP. We will test the hypothesis that the most important nucleoprotein barrier to replication (RNAP) recruits the means (UvrD) of resolving conflicts between transcription and replication. This work will establish why gene expression poses such a problem for genome duplication, how cells minimise the conflicts between two such fundamental processes and the possible evolutionary constraints on all organisms with respect to simultaneous transcription and DNA replication.

DNA replication, transcription and DNA repair are all important targets for cytotoxic agents and so one key beneficiary of this work will be pharmaceutical companies. Our analysis of these processes may aid therefore the development of antimicrobial and anti-cancer agents. Additionally, unresolved blocks to replication are either lethal or contribute to potentially catastrophic genome rearrangements. Many companies have developed therapeutic and/or diagnostic reagents related to genome instability and human health, and an increased understanding of how transcription contributes to replicative blockage, and how cells resolve this conflict, will increase the knowledge base needed for continued development. The emerging field of synthetic biology will also benefit from this project. Development of partially or fully synthetic cells requires the ability both to replicate the cell and to maintain gene expression. Any synthetic system must therefore either avoid conflicts between genome duplication and gene expression, or minimise such conflicts. This project is aimed at understanding the reasons why gene expression presents a major problem for genome duplication, and how evolution has resulted in natural systems that minimise this problem. The work will therefore inform the efforts of synthetic biologists to develop self-replicating systems that can sustain effective gene expression. Members of the public will also benefit from this proposed research. Understanding the interplay between genome duplication and gene expression may underpin development of novel pharmaceuticals with respect to human, animal and crop health. This project will also contribute to the design of synthetic organisms with potential benefits for energy production, bioremediation or pharmaceutical synthesis. This study will therefore contribute to the economic, health and environmental quality of life of those living in the UK. Increased understanding of how genome duplication, gene expression and genetic stability are linked will also contribute to the public understanding of healthcare issues related to genomic instability. The work described here will also contribute to the generation of a scientifically-literate workforce not only via training of the research assistants employed on this project but also by contributing to a research-led environment for the teaching of undergraduate students. The Research and Innovation Unit at the University of Aberdeen and the Research and Enterprise Development Unit at the University of Bristol both have extensive experience in the identification of research with potential for commercial exploitation and the protection of intellectual property. Both units also have multiple contacts with biotechnology and pharmaceutical companies. These links will be exploited if findings resulting from this proposed research are assessed as having any commercial potential for development. Additionally, NS and MD are both members of the joint research council-funded 'Synthetic Components Network'. Interactions with this network will therefore ensure wide dissemination of our results to the synthetic biology field and may also lead to collaborative links with synthetic biologists. Communication of data arising from this project will not therefore rely solely on publication in peer-reviewed journals or presentations at specialised scientific meetings. We will also communicate our findings to the general public, aided by dedicated communication teams at the universities of both Aberdeen and Bristol. All research papers will be publicised on our websites. Additionally, key research findings that may be of general interest to the public will be communicated to both local and national media outlets via press releases. However, established media outlets will also be circumvented to provide more direct communication to the public by participation in public engagement with science events both in Bristol and in Aberdeen.