Modelling genomic instability in Drosophila melanogaster: genetic and molecular analysis of Dm WRN exonuclease
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
Some people age well while other succumb to many and various diseases associated with old age including cancer, diabetes and heart disease, but we still don't know why some people age more healthily than others. By studying a human premature ageing disease called Werner's Syndrome which essentially mimics normal ageing but much more quickly, scientists hope to understand the causes of many of the age-associated diseases. It is now known that most, if not all, of the many premature ageing aspects of Werner's syndrome result from the loss of a single protein known as WRN. We have shown that cells taken from patients with Werner's syndrome have problems copying their genetic blueprint, the DNA, and that such problems result in their DNA becoming very unstable. Such 'genomic instability' is known to be important in the development of many cancers, and it may also make people more prone to develop other serious diseases. To find out how WRN has such wide-ranging effects, we need to study the protein on its own in the test tube and to test the effect of making changes in it (mutations) in cells and whole organisms. Although some progress has been made using cells taken from patients, each batch of cells is different and we cannot precisely ascribe any changes to the WRN protein itself. In addition, we need to work out which parts of the WRN molecule are particularly important in keeping the DNA intact, and whether changes to these regions can affect how long a cell and even an organism lives. Obviously this is both impractical and unethical in mammalian systems, so we have chosen to examine the fruit fly, Drosophila. The fly is very well characterised, with the sequence of all the 'letters' of its DNA code already known, and many different genetic procedures are possible to 'mix and match' different combinations of genes. To date, we are the only researchers to have identified a WRN-like function in flies and we have very recently, and excitingly, shown that flies lacking this activity show a big increase in abnormal changes to their DNA, just like in patients with Werner's syndrome. By providing a full molecular and genetic analysis of the action of WRN in flies, we hope to answer significant questions as to what the protein is doing to protect our DNA. We are therefore planning to make molecular probes to look at when and where the fly WRN protein is made, what it does to DNA, what other proteins it works with and what aspects of the DNA copying process are affected when the protein is altered. Finally, by replacing the fly version of the WRN gene with the human WRN gene, we hope to develop a model system for future use in testing drugs that either block human WRN activity (for example to make otherwise long-lived cancer cells age quickly), or to promote longevity of normal cells, with the hope of avoiding many of the problems associated with ageing of vitally important cells in old age. The long term aim of such ageing research, and in particular, the development of model ageing systems, is to help achieve a healthy old age and prevent many of the debilitating illnesses associated with getting old.
Technical Summary
Werner's Syndrome is very useful model of ageing. Loss of function of the unique WRN exonuclease/helicase leads to pleiotropic effects including increased genomic instability, which may arise from illegitimate recombination at stalled replication forks. We have identified a homologue of the WRN exonuclease in Drosophila, and shown that mutation of this gene results in a large increase in genomic instability. We propose to identify the processes of DNA metabolism for which DmWRNexo is required through parallel genetic, cell biological and biochemical studies of DNA replication, recombination and repair. DmWRNexo mutation effects will be analysed phenotypically through development and in the post-mitotic adult. Using RT-PCR, novel antibodies and tagged gene variants, we shall assess DmWRNexo spatial and temporal expression through development and the cell cycle. Sensitivity of WRNexo mutant flies to DNA damaging agents will be examined. We shall characterise the biochemical activities of the DmWRNexo in terms of DNA substrate preferences, its impact on stalled replication fork resolution, and roles in recombination and DNA repair. We propose to identify protein partners of DmWRNexo, in particular examining the known RecQ helicases in Drosophila. Complementation of the genetic instability phenotype by human WRN transgenes will be tested, providing the possibility of a 'humanised' model system as a powerful tool for the analysis of agents that impact on ageing through the WRN pathway, whilst avoiding use of mammalian models. The role of Holliday junction persistence in genomic instability in DmWRNexo null flies will be tested by introducing RusA, a bacterial HJ resolvase, under the control of the WRNexo promoter. The data generated should provide exciting insights into the roles of the exonuclease activity of WRN distinct from its helicase activity. The contribution of genomic instability to accelerated ageing may also be made possible by this DmWRNexo mutant fly.
Organisations
People |
ORCID iD |
Lynne Cox (Principal Investigator) |
Publications
Bird JL
(2012)
Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.
in Biogerontology
Boubriak I
(2009)
DmWRNexo is a 3'-5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease.
in Biogerontology
Cox L
(2007)
From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing
in Cellular and Molecular Life Sciences
Cox L S
(2012)
Encyclopedia of DNA research
Cox LS
(2007)
Modeling Werner Syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease.
in Annals of the New York Academy of Sciences
Cox LS
(2009)
Cell senescence: the future of ageing?
in Biogerontology
Cox LS
(2009)
Live fast, die young: new lessons in mammalian longevity.
in Rejuvenation research
Cox LS; Boubriak I
(2010)
DNA damage, repair mechansims and ageing
Description | We have identified the fly version of a human gene important in ageing. Using the fly system, we were able to understand more clearly how the encoded protein protects the DNA from damage as cell divide and how its loss may contribute to ageing. We have developed new ways of looking at how the protein works and hope to be able to use these technologies to test agents that can be used either to enhance its activity (anti-ageing) or inhibit it (anti-cancer). We also discovered by chance a very similar gene that seems to be important in protecting cells from another type of DNA damage and mutation of which in humans is linked to a familial cancer syndrome. |
Exploitation Route | The exonuclease assay may be adaptable for higher throughput drug screening. The key findings on DmWRNexo activity have been published in a number academic journals (inlcuding a video journal to show exactly how we conduct the novel exonuclease assay) , and presented at conferences and to funders. The novel gene discovery linked in with our findings on BB/E016995/1 and have formed the basis of a several grant applications for further funding, together with a PhD project at the Open University. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | Our findings have been used by BBSRC to highlight the value of basic biosciences underpinning health, featuring in their online 20 year timeline and in print material at the recent celebration of British Biosciences. |
First Year Of Impact | 2009 |
Sector | Other |
Impact Types | Cultural |
Description | Age UK studentship - DmWRNexo |
Amount | £79,000 (GBP) |
Organisation | Age UK |
Department | Research into Ageing Fund (RiAF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2008 |
End | 09/2011 |
Description | Age UK studentship - a worm model of Werner syndrome |
Amount | £75,000 (GBP) |
Organisation | Age UK |
Department | Research into Ageing Fund (RiAF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2010 |
End | 09/2013 |
Description | BBSRC Responsive mode |
Amount | £473,213 (GBP) |
Funding ID | BB/M006727/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2015 |
End | 02/2018 |
Description | BBSRC responsive mode |
Amount | £522,415 (GBP) |
Funding ID | BB/M006727/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2015 |
End | 12/2017 |
Title | Drosophila model for analysis of the exonuclease activity of WRN |
Description | Transgenic fly bearing piggyBac mutation in 5' UTR of CG7670 locus: we did not create the fly stock, but have now characterised it phenotypically in terms of WRN exo expression through development, DNA instability and drug sensitivity. Several totally novel fly strains were also generated on this project including flies with: (i) a point mutation in CG7670 altering D229 to valine (ii) D229V/ pBac (iii) pBac/deficiency (iv) pBac/pBac and mus309 (DmBLM) double mutant (v) RFP-t We have further characterized a novel fly strain with mutation in a related gene CG6744; this work highlighted its potential as a component of the Fanconi anaemia pathway and was cited in Molecular Cell by a group who are working on the human homologue. |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Provided To Others? | No |
Impact | We have lodged all our findings including new gene sequence information in Genbank and Flybase, contributing to the knowledge base and providing annotated function for a previously unannotated gene. |
Title | Monospecific antibodies against human WRN and DmWRNexo |
Description | Rabbit polyclonal antibody against short peptide of human WRN (KLH-coupled); similar antibody generated against DmWRNexo |
Type Of Material | Antibody |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | Supplied anti-DmWRNexo Ab to collaborator for use in DPhil project - paper reporting immunofluorescence staining of DmWRNexo during embryonic development is nearly ready for submission for publication. |
Description | CSH conference on Aging 2010: Loss of Drosophila Werner exonuclease leads to replication defects and failure of chromatid segregation during early development |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | R S (co-PI on the grant) was an invited speaker at the Cold Spring Harbor aging meeting. We co-wrote the talk and have published much of the data her discussed in the peer-reviewed literature (see Publications). We continue to collaborate and are now preparing a new responsive mode grant proposal for work arising from the original study. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | Human ageing gene found in flies - showcase at UK Funders' Forum |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Showcasing of our research by the BBSRC at UK Funders' Forum Printed material describing our research was disseminated and we answered direct questions from participants. Our work has subesequently been included as a highlight in BBSRC's 20 years of Bioscience research on their web page and was included in a small booklet given out to delegates at a major BBSRC event early 2014 |
Year(s) Of Engagement Activity | 2008,2009 |
Description | Human ageing gene found in flies - TV and radio interviews |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Media (as a channel to the public) |
Results and Impact | TV and radio interviews based on a press release of our findings published in Aging Cell. BBC Oxford news TV interview about our discovery of Drosophila WRN exonuclease and its implications for research on ageing. Radio Oxford interview about our discovery of Drosophila WRN exonuclease and its implications for research on ageing. Unsure |
Year(s) Of Engagement Activity | 2008 |
Description | Human ageing gene found in flies - YouTube video |
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 | You Tube video produced by BBSRC Media for dissemination of our research findings Large uptake by UK print press and many international news agencies. Invitations to give radio and TV broadcasts. |
Year(s) Of Engagement Activity | 2008,2009 |
URL | http://www.youtube.com/watch?v=IGQwz2Wv9FU |
Description | IABG conference 2007: Modelling Werner syndrome in the fruit fly |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Type Of Presentation | Paper Presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Conference presentation 12th Congress of the International Association of Biomedical Gerontology (IABG). Outcome: paper published in Annals of the New York Academy of Sciences. Further collaboration planned with Dr Robert Saunders, Open University. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2007 |
Description | International Science Festival, Edinburgh, 2015 (L. Cox) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I was a member of an expert panel of three; each of us gave a talk (mine was on progeroid diseases and ageing research), followed by a open discussion in which panel members answered audience questions. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.physoc.org/press-release/2015/forever-young-can-we-cure-ageing-%E2%80%93-edinburgh-intern... |
Description | Invited keynote at Biopharma meeting, UCL 23rd Feb 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Undergraduate students |
Results and Impact | I gave a 1 hour invited presentation on prospects for developing new medicines for age-related diseases through understanding cellular senescence, and then sat on a discussion panel (with 2 other scientists) to take audience questions on the entire workshop. |
Year(s) Of Engagement Activity | 2019 |
Description | Modelling genomic instability of Werner's syndrome in the fruit fly; BSRA 2007 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited speaker at British Society for Research on Ageing annual scientific meeting, Liverpool 2007 Unsure |
Year(s) Of Engagement Activity | 2007 |
Description | OxAgeN - establishment of the Oxford Aging Network (L. Cox) |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Established a network of scientists and clinicians with an interest in ageing (research and clinical practice) to foster closer collaboration and accelerate discoveries from bench to clinic. Representatives from commercial organisations and third sector (U3A, Age UK) are also members. Impact: decision to hold scientific conference on ageing in Oxford. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.imm.ox.ac.uk/ageing |
Description | Talk, MitOX Dec 2015 (L Cox) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Invited talk at mitochondrial conference on a WRN-related protein in mitochondrial DNA maintenance. outcome: request to publish sceintifc research paper in Biochemical Society Transactions. |
Year(s) Of Engagement Activity | 2015 |
Description | The role of WRN in cellular senescence - ageing workshop 2006 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Invited speaker at international ageing workshop, Bramber, Brighton increased visibilty of my lab's research. Subsequent discussions and research collaboration initiated with collegaues at the meeting |
Year(s) Of Engagement Activity | Pre-2006,2006 |