Award details

Recruitment of DNA repair enzymes to stalled transcription complexes

Reference	BB/E004695/1
Principal Investigator / Supervisor	Professor Nigel Savery
Co-Investigators / Co-Supervisors
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	270,043
Status	Completed
Type	Research Grant
Start date	26/02/2007
End date	25/04/2010
Duration	38 months

Abstract

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.

Summary

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 copiedhave 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.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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