Recruitment of DNA repair enzymes to stalled transcription complexes

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

Mechanisms for repairing damaged DNA are essential for the survival of all forms of life. DNA molecules are long strands made up of four different types of small molecule, called nucleotides. The order in which these four types of nucleotide are arranged along the DNA carries many different types of information: genes are sequences of nucleotides that contain the information necessary for making a particular types of protein; some other sequences of nucleotides are 'switches' that control when and where particular genes are turned on; other sequences give instructions about making a new copy of the DNA molecule, and so on. The DNA in every cell is under constant attack, from the ultraviolet radiation in sunlight, from mutagenic chemicals in the environment, from ionising radiation, and even from the by-products of the cell's own metabolism. Chemical modification of the nucleotides within DNA can change or destroy the information that they carry, and without mechanisms for repairing damaged sections of DNA the information within a cell's genome would rapidly become so corrupted that the cell would be unable to function. A variety of DNA repair systems have evolved to counteract the constant damaging of DNA. Most of these use a combination of enzymes to bind to the damaged nucleotide(s), cut out the damaged section of DNA and replace it with a 'patch' of new nucleotides. The system that is used to repair damaged DNA depends on both the type of damage that has occurred, and the type of DNA that it occurs in. The repair enzymes face particular difficulties when DNA damage occurs in a gene that is turned on. In order for the information in a gene to be used by the cell a messenger RNA copy of the gene must be made by an enzyme called RNA polymerase, in a process called transcription. RNA polymerase separates the two strands of the double-helical DNA molecule, and uses one strand as a template for the synthesis of messenger RNA. If nucleotides on the strand being copied have been damaged, RNA polymerase can become jammed. The gene cannot be transcribed until the damage is repaired, but because the DNA damage is lodged within RNA polymerase the DNA repair enzymes cannot reach it to repair it. This is potentially lethal for the cell. Specialised proteins called transcription-repair coupling factors overcome this problem, and as a result DNA damage that blocks RNA polymerase in active genes is repaired more quickly than DNA damage in other regions of DNA. The purpose of this research is to understand how a transcription-repair coupling factor speeds up the rate of DNA repair in active genes. This project utilises RNA polymerase and repair enzymes from a bacterial model system. We have previously investigated how this transcription-repair coupling factor removes the jammed RNA polymerase from the damaged DNA. In this work we will investigate how the interactions between the transcription-repair coupling factor and the DNA repair proteins lead to damage being repaired more quickly when it is encountered by RNA polymerase than it is when the repair proteins function on their own. The lessons learnt in this model system will contribute to our understanding of the way that similar proteins may function in more complex organisms. Proteins that manipulate DNA play critical roles in many fundamental cellular processes and also have important applications in biotechnology. By gaining a thorough understanding of the ways in which such proteins function we aim to contribute to increased understanding of disease and the design of novel therapeutic strategies.

Technical Summary

The E. coli Mfd protein is a transcription-repair coupling factor that is responsible for prioritising the repair of DNA lesions within transcribed genes. Recent work has focused on determining how Mfd displaces transcription complexes stalled by DNA damage. The aim of this proposal is to learn how Mfd recruits the UvrAB(C) DNA repair apparatus and promotes an increased rate of repair. Our experimental approach will be to generate mutant proteins defective in single specified activities by a combination of site-directed mutagenesis and bacterial genetics. The properties of the wild-type and mutant proteins will be studied in a range of in vitro assays, and their ability to support transcription coupled repair will be assessed in vitro and in vivo. Our objectives are: (1) To define the surfaces of Mfd and UvrA that interact with one another, and to understand the interplay between UvrA:Mfd interactions and UvrA:UvrB interaction. Parts of Mfd and UvrB are homologous, and it is likely that they compete for sites on UvrA during repair complex assembly. (2) To determine the role of the C-terminal domain of Mfd in controlling Mfd:UvrA interactions. The crystal structure of Mfd revealed that the C-terminal domain occludes the likely UvrA contact surface, and the properties of an Mfd derivative lacking this domain support the theory that it may have a regulatory function. (3) To define the properties of Mfd and UvrA that are required for damage recognition and repair-complex assembly during transcription-coupled repair. Both Mfd and UvrA are multifunctional proteins, and in order to understand their roles in transcription-coupled repair we must determine which activities are essential in that context and which are redundant. (4) To define the boundaries of Mfd's zone of influence around stalled transcription complexes. To understand the mechanism by which Mfd directs repair proteins to DNA lesions it is important to know the limits of its ability to do so.
 
Description From grant final report, submitted 2010.

We have shown that:
1. bacterial TCR bypasses the need for DNA damage-recognition by UvrA, and the "insertion domain" of UvrA is involved in damage-recognition. This defines the point at which Mfd acts, and identifies new similarities between prokaryotic and eukaryotic TCR
.
2. the UvrB-homology module of Mfd is responsible for strand-specific repair, that residues within it that interact with UvrA, and that this module forms part of a "clamp" that autoregulates Mfd activity and is held shut by interdomain contacts with the C-terminal domain.

3. stalled transcription complexes can affect DNA repair efficiency at a distance. This overturns the paradigm for damage recognition in TCR, and suggests a model in which Uvr proteins are loaded onto DNA in a strand-specific manner and DNA translocation occurs after RNA polymerase is displaced.

We investigated the molecular mechanisms of transcription-coupled DNA repair (TCR), a pathway that ensures that when DNA within an active gene is damaged it is repaired more quickly than similar damage in other regions of the genome. TCR is involved in the generation of antibiotic-resistant mutants of some types of bacteria, and a similar pathway is important for reducing the number of potentially cancer-generating mutations within human tissues. Our work centred on trying to understand how TCR is accelerated relative to the repair in other parts of the genome.
At the start of the project the identities of the proteins involved in TCR were known, but it was not clear how they interacted with one another. A large part of our work involved making specific alterations to the proteins that were involved in the process, and then testing the effects of these changes on normal repair and TCR in cells and in systems built from purified components. The most important finding from these experiments was that the very first step of the repair pathway, the way in which the damaged DNA was recognised, differs between TCR and normal repair. We found that 3 different alterations to the DNA repair protein UvrA affected normal repair more than they affected TCR. All 3 disrupted the ability of the protein to distinguish between damaged and undamaged DNA (in the case of one of them this had not been shown before, and so our work identified a new function for one region of UvrA). This indicates that the Mfd protein (a transcription-repair coupling factor that is not required for normal repair), together with RNA polymerase (the machinery that makes RNA copies of genes when that gene is turned on), negates the need for damage recognition by UvrA during TCR, and distinguishes between 2 models that have been proposed in the literature. Our work with altered proteins also allowed us to identify how several of the proteins involved in the process interact with each other, and to examine the way in which these interactions regulated the activities of the protein concerned. For example; we found that the ends of the Mfd protein interact with one another to form a "clamp" that keeps the protein inactive until it interacts with other proteins, we showed how Mfd interacts with UvrA, and we showed that this interaction was essential for the acceleration of DNA repair.
TCR is triggered when a DNA lesion causes RNA polymerase to stall, and as only lesions in one of the two strands of the DNA double helix are able to do this TCR only affects one strand. Before this work began it was felt likely that on the rare occasions that a lesion was present on the other strand close to a stalled RNA polymerase it would also be subject to TCR: in other words a stalled RNA polymerase would attract repair enzymes, and anything within the vicinity would be repaired quickly. We devised an assay that allowed us to test this prediction, and we found that repair of the DNA flanking a stalled RNA polymerase was strand specific: a stalled RNA polymerase (together with Mfd) can accelerate the repair of lesions several hundred base pairs away, but it always accelerates the repair of just one strand. This indicates that the loading of the repair enzymes is not random, but is strand-specific, and that during the search for DNA damage a complex of proteins must move along the DNA until the lesion is reached.
Taken together, our results have led to a far better understanding of the molecular basis of a well-conserved pathway that is important for maintaining the integrity of the genetic message as it passes between generations. The long term impact of fundamental research such as this cannot be predicted with accuracy, but in the short to mid-term we suggest that this information is likely to be applied by researchers looking for ways to inhibit the production of antibiotic-resistant mutant bacteria, and synthetic biologists involved in the design of genomes for engineered bacteria.
Exploitation Route The research project concerned a mechanism for the maintenance of genome stability in bacteria. Consequently, our results are of use to researchers studying processes arising from genome instability (such as mutation giving rise to antibiotic resistance, or altered properties of pathogenic or commercially important bacteria), and to researchers and industrial labs aiming to engineer, or design from scratch, the genomes of cells to reliably perform specific functions (such as the production of high value chemicals). Since the research project was completed it became apparent that the protein at the centre of our research was, counter-intuitively, involved in promoting mutation to antibiotic resistance in the important food-borne pathogen Campylobacter jejunii. Our discoveries about how the protein works are helpful in thinking about how to reduce the rise of antibiotic resistance in this organism.
Sectors Agriculture, Food and Drink,Education,Manufacturing, including Industrial Biotechology,Other
 
Description This research project related to the understanding of a fundamental process in biochemistry. It is expected that most short term impact of this research will relate to its influence on the work of other academic laboratories, which are not considered in this section. In the long term this research is anticipated to have direct or indirect impact on a number of fields outside academia (see RCUK Key Findings), but the measurable societal impact at present has come through public engagement activities (Open days, Science cafe)
First Year Of Impact 2007
Sector Education
Impact Types Cultural,Societal
 
Description Press release (Manelyte paper) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Not monitored.
Year(s) Of Engagement Activity 2010
URL http://www.bristol.ac.uk/news/2010/7382.html
 
Description University of Bristol Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact One-to-one discussions with members of the public about our research, biochemical sciences generally, and other aspects of University study.

It is difficult to report specific impacts. I have no doubt that the discussions influenced the life choices of many of the young people that I spoke with.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015