Determining the Molecular Mechanism of Global Genome Repair in Saccharomyces cerevisiae

Lead Research Organisation: University of Wales
Department Name: UNLISTED

Abstract

All forms of life, including humans, contain within each of their cells the encrypted information necessary for coordinating the function of individual cells and the overall organization of the organism. The information is stored in the nucleus of the cell within a macromolecule called deoxyribonucleic acid - DNA. This information is divided into units called genes, the sum of all the genetic material of an organism is referred to as its genome. Life?s coded information might be expected to be extremely stable and resistant to change, since altering the coded message could have serious consequences. On the other hand, organisms are able to adapt to changes in their environment by virtue of the genetic variation within the population caused by alterations in the genetic material of each individual. This process is known as evolution. Much of the genetic variation within the human population is the result of ?reshuffling? the genes during sexual reproduction ? a process known as recombination. However, DNA can also be altered by normal processes operating within the cell, and physical or chemical damage from the environment, including ultraviolet radiation from sunlight. Each of our cells receives around 10,000 DNA lesions each day. If left unchecked this damage would quickly erode the genetic material, since replication of damaged DNA during cell division can permanently alter the genetic code. Consequently a number of different DNA repair pathways have evolved over time. Collectively these are fundamental to the stability of the genome. People who inherit defects in the genes controlling these DNA repair pathways are more likely to suffer from certain cancers. Our research aims to understand how one of these processes, nucleotide excision repair [NER] operates. People with defects in genes regulating this process suffer highly elevated levels of skin cancer. Molecular studies have revealed how defects in different genes involved in the process can result in three distinct diseases. Much of our knowledge has come from the study of NER in a variety of different organisms. We study NER in bakers? yeast - amazingly, the process is remarkably similar to that in human cells. We recently identified a novel stage in the mechanism of NER in yeast. If this mechanism exists in human cells, it could have important implications for the use certain types of cancer treatments.

Technical Summary

The repair of damaged DNA is fundamental to the maintenance of genome integrity. Genetic predisposition to cancer in somatic cells can be inherited due to malfunction of genes required for the normal processing of DNA damage by three major DNA repair pathways including nucleotide excision repair. Defective NER is associated with the hereditary cancer-prone disease xeroderma pigmentosum (XP). The frequent association of the XP homozygous state with various types of skin cancers demonstrated the importance of NER as a fundamental mechanism for protecting the functional integrity of the human genome, and provided important confirmation of the somatic mutation theory of cancer. In addition, NER contributes to the normal development of the organism including the formation of the immune system and aging. DNA damage can be caused by exposure to chemicals or electromagnetic radiation including UV from sunlight. NER repairs a wide spectrum of DNA lesions and has a complex biochemical mechanism. Two damage-recognition pathways are suggested to operate during NER. The transcription coupled repair pathway [TCR] that operates on the transcribed strand of active genes, and the global genome repair pathway [GGR] that operates in non-transcribed DNA. Our research investigates the molecular mechanism of GGR. We study this process in Saccharomyces cerevisiae. The NER process in yeast cells is remarkably similar to that in human cells. We have isolated a complex of yeast proteins that is required for GGR function. This complex generates supercoiling in damaged DNA, and this is necessary for the excision of DNA damage during GGR. One component of this project will use a combination of established molecular techniques and a novel physical technique to analyze the mechanism of supercoil formation. Much of our understanding of the biochemistry of NER is derived from studies using nucleosome free DNA templates. Preparation of yeast extracts that support NER on nucleosome-free DNA do not support NER on nucleosome-assembled DNA. We have recovered a chromatin associated fraction that is lost during extract processing, which recovers the NER activity on nucleosomal templates. We will purify and characterize this activity to determine its function in NER. Finally, we previously identified a non-proteolytic role of the 19S proteasome in NER. We have recently identified the E3 ubiquitin ligase that regulates this function in yeast. We will now examine the molecular architecture of this ligase to examine how it is regulated in response to DNA damage and determine its mechanism in NER

Publications

10 25 50
 
Description Chairman of UK Genome Stability Network
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact Provides a forum for training and development of UK based students and post docs working in the field of genome stability.
 
Description EEMS Committee member
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description Training of UK based genetic toxicologists
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Continuous development for professional genetic toxicologists in UK industry.
 
Description Working Group for Improved Radiation Therapy
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact The working group aims to improve the delivery of radiotherapy in the UK.
 
Description BBSRC CASE studentship
Amount £150,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description CRUK Project Grant
Amount £250,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description Commonwealth Scholarship and Fellowship Plan
Amount £114,400 (GBP)
Organisation Government of the UK 
Department Commonwealth Scholarship Commission
Sector Public
Country United Kingdom
Start 01/2015 
End 01/2018
 
Description Feasibility Grant (Innovate UK/Unilever)
Amount £75,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 03/2015 
End 03/2016
 
Description Feasibility grant
Amount £240,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2015
 
Description Infrastructure Award to Establish Structural Biology as a Discipline at Cardiff
Amount £60,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 09/2010
 
Description Knowledge Transfer Partnership Award
Amount £250,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 12/2011 
End 07/2013
 
Description MRC Project Grant
Amount £450,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2012 
End 02/2016
 
Description MRC Studentship (Determining the non-proteolytic function of a novel E3 ubiquitin ligase in NER)
Amount £26,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2006 
End 09/2009
 
Description MRC Studentship (Investigating the role of ABF1 binding in nucleotide excision repair)
Amount £53,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 09/2010
 
Description NISCHR Cancer Genetics BRU Award
Amount £1,500,000 (GBP)
Organisation Health and Care Research Wales 
Sector Public
Country United Kingdom
Start 04/2011 
End 03/2014
 
Description Studentship (BBSRC/AstraZeneca)
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2020
 
Description University of Cardiff College of Medicine, Lord Merthyr Scholarship (Integrative analysis of chip-on-chip datasets in Saccharomyces cerevisiae)
Amount £30,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 01/2008 
End 12/2011
 
Description VIP Award
Amount £35,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2006 
End 09/2007
 
Title Expression arrays for UV induced gene transcription 
Description Gene expression profiles for UV regulated genes by an novel E3 ubiquitin ligase 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact EMBO J 2006 paper and PhD thesis 2010. 
 
Title GGNER Complex Function In Vivo 
Description Novel strains to investigate function of the GGNER proteins in yeast. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact Papers JBC - 18996839 EMBO J -16675952 
 
Title GGNER Complex Purification and Characterisation 
Description Newly identified E3 ligase function of the GGNER complex. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2007 
Provided To Others? Yes  
Impact Papers EMBO J - 16675952 JBC - 18996839 
 
Title Omics method for detection of DNA damage and repair 
Description Novel tools for detecting genome wide DNA damage and repair at high resolution throughout genomes 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact NAR paper 2010 IP published 2009 Collaboration with Agilent Technologies and successful applications for KTP to develop the method for gentox testing and NISCHR BRU grant awarded. 
 
Title Tools for analysing chip chip data for DNA damage induction 
Description Bioinformatic tools for normalising and peak calling for the detection of UV induced DNA damage by Chip CHip. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact NAR paper 2010 
 
Title Yeast Rad4 antibody, Yeast Rad33 antibody 
Description Novel antibodies against two key NER proteins 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact EMBO J paper on Rad23/4 function in NER 
 
Description Biochemical Function of the Yeast GGNER Complex 
Organisation University of Dundee
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We uncovered the biochemical activities of the yeast GGNER complex in chromatin remodelling.
Collaborator Contribution Our collaborator, Tom Owen-Hughes trained Dr Shirong Yu who is funded by this award at Dundee in several key chromatin remodelling assays.
Impact JBC paper - find pub med ref Owen-Hughes wrote a review in which this work was cited.
 
Description Generating Antibodies agaist Novel NER Components 
Organisation Enzo Life Sciences
Country United Kingdom 
Sector Private 
PI Contribution We identified a number of novel NER factors and are using the newly generated antibodies to discover the functions of these proteins.
Collaborator Contribution Enzo Life Sciences have generated a number of key peptide antibodies raised against several new NER factors.
Impact EMBO J Paper - 16675952 JBC Paper - 18996839
 
Description Macromolecular Machines - Proteasome Structure 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided purified protein complexes for structural analysis by consortium members.
Collaborator Contribution Provides structural information regarding the role of the 19S proteasome during nucleotide excision repair.
Impact Consortium bid to EU submitted December 2009.
Start Year 2009
 
Description Studies on Global Genome Repair in Yeast 
Organisation University of California, San Diego (UCSD)
Department Department of Pathology
Country United States 
Sector Academic/University 
PI Contribution My research demonstrated specific biochemical activities of the GGNER complex which were then investigated further by examining in vivo DNA repair assays.
Collaborator Contribution Certain DNA repair assays for interpretation of specific biochemical data.
Impact EACR Cancer Researcher Award International Patent granted Publications 18996839 17150417 16675952
 
Description The Role of the Proteasome in Nucleotide Excision Repair 
Organisation University of Arizona
Department Center for Innovations in Medicine
Country United States 
Sector Academic/University 
PI Contribution We provided all of the expertise and reagents necessary for investigating the connection with nucleotide excision repair.
Collaborator Contribution Our collaborator, Stephen Johnson provided a number of key strains and reagents necessary for our investigation into the role of the proteasome in NER.
Impact EMBO J - 16675952 Roads to Rome review - 17150417
 
Title Genome wide assay for measuring DNA damage 
Description Genome wide assay for measuring DNA damage 
IP Reference WO2009001111 
Protection Patent granted
Year Protection Granted 2008
Licensed No
Impact Partnership with Agilent technologies
 
Title Genomic tools for measuring DNA damage 
Description Using Chip-chip/Chip-Seq methods to detect DNA damage [and repair] throughout genomes. Is being applied to genotoxicity testing and patient stratification of chemo drugs. Funded by: KTP [TSB/Agilent Technologies] NISCHR Cancer Genetics BRU CRUK Centre Clinical PHD fellow BBSRC/GSK Case award 
Type Support Tool - For Medical Intervention
Current Stage Of Development Initial development
Year Development Stage Completed 2012
Development Status Under active development/distribution
Impact Still being developed for different applications. Genotoxicity testing/stratified medicine 
 
Description Conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentations at national and International conferences on DNA Repair and Genome Stability and Genetic Toxicology in Holland, Denmark, Cambridge, France, Prague.

Webinars for Agilent Technologies and Thomson Reuters - International coverage
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL http://stateofinnovation.com/researcher-highlight-series-simon-reed?word=reed
 
Description Public Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Public lectures to local groups including various charity organisations, eg Rotary Club.

Donations to Cancer Research Wales.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Schools
Results and Impact Careers talks and lab visits recruitment of 6th formers to Cardiff University.

lab visit
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Website - Personal Research 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Publushed on line lay information for research undertaken in the group.

Invitations to speak at a number of charity fund raising events including Rotary Club, Cancer Research Wales and Probus.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010