Studies on the organelle-specific functions of human Type 1A topoisomerase TOP3A

Lead Research Organisation: University of Lincoln
Department Name: School of Life Sciences

Abstract

Mitochondrial DNA depletion syndromes are a group of clinically heterogeneous genetic diseases that affect patients in infancy and childhood. Hitherto nine genes have been identified to be associated to these diseases, however, there are a considerable number of verified cases, where the mutated gene responsible for the disease has not been identified. The basic science research project proposed in this application will considerably enhance our understanding of the molecular mechanisms responsible for mitochondrial DNA depletion syndromes, and open up further avenues for the treatment of its sufferers.

In most living organisms biomaterial that builds the body of cells is coded in the DNA. This coded information is read in a process termed RNA transcription. Before the division of cells, in order to maintain the equal distribution of DNA content between the daughter cells, the DNA has to be duplicated; this process is termed DNA replication. During these rather complicated processes, the DNA has to open up to give access to enzymes that function in DNA metabolism. Separation of the two strands of DNA is catalysed by helicase enzymes. During the unwinding of the DNA double helix by the helicase enzymes, torsional stress is generated, which is relieved by another set of enzymes called topoisomerases. Topoisomerases are also capable of tidying up tangled stretches of DNA.

The powerplant of cells, the mitochondria, also contain DNA, but - curiously enough - this DNA is circular like DNA of most bacterial cells, and unlike the linear chromosomal DNA that can be found in the nucleus of eukaryotes. Mitochondrial DNA is more susceptible to damage than nuclear DNA because of the metabolic processes that take place in the mitochondria.

One of the enzymes that guard the integrity of nuclear as well as mitochondrial DNA is DNA topoisomerase III alpha (TOP3A). The complete lack of this enzyme in mice causes lethality during early embryonic development, while its reduced levels in cell culture cause the accelerated ageing of cells. Though the biochemical activities of TOP3A have been well characterised, its exact function is largely unknown, and even less is known about its role in maintaining the integrity of the mitochondrial genome.

In my preliminary work I have set up a system that allows the depletion of TOP3A from the cells, while specific expression and function can be maintained in either the nuclei or in the mitochondria, exclusively. This system permits the analysis of nuclear and mitochondrial functions of TOP3A independently of each other to reveal why the ageing process is accelerated in cells devoid of TOP3A, and whether it can be attributed to a function of the enzyme specific to the nuclei or the mitochondria. More specifically, we will investigate how the replication of mitochondrial DNA, and the metabolic activity of mitochondria are affected in cells that lack mitochondrial TOP3A function. We will study how suppression of nuclear TOP3A functions affects nuclear DNA metabolism, chromosome segregation and cell divisions. We will also identify and characterise protein partners of TOP3A that assist its organelle-specific functions.

Technical Summary

Despite the recent advances in the biochemistry of TOP3A, it is not clear what processes it participates in the nucleus and what its role is in the maintenance of mitochondrial DNA (mtDNA). The phenotypic consequences of TOP3A depletion are also rather complex and difficult to attribute to either the nuclear or mitochondrial role of the enzyme.

In this proposal we will dissect the essential cellular role of TOP3A in maintaining the integrity of nuclear and mitochondrial DNA. In my preliminary work I have set up a system that permits the selective expression of either the nuclear or the mitochondrial form of TOP3A in the background of its systemic RNAi-mediated depletion. We will use this system to verify the phenotypic consequences of TOP3A depletion and to attribute each of the phenotypes to either the mitochondrial or the nuclear function of the enzyme. These experiments will be based on microscopic observations following staining with specific antibodies or the senescence marker beta-galactosidase.

We will study how elimination of TOP3A from the mitochondria affects mtDNA replication and mitochondrial metabolic activity. We will use two-dimensional agarose gel electrophoresis (N2D-AGE) to visualise replication intermediates and other DNA structures and quantitative real-time PCR to measure mtDNA copy number. Metabolic activity of mitochondria will be monitored with measuring ATP and ROS concentrations and mitochondrial membrane potential.

We will map the protein interaction network of TOP3A both in the mitochondria and in the nucleus. GFP tagged mitochondrial or nuclear forms of TOP3A will be expressed in cells and the tagged protein will be specifically pulled down using the GFP-nanotrap technology. Proteins specifically bound to TOP3A will be identified by mass spectrometry. The interaction will be verified with in vitro (co-immunoprecipitation, far-Western), and microscopy-based cellular techniques (FRET, bimolecular fluorescent complementation).

Planned Impact

Loss of mitochondrial DNA or damage to mtDNA leads to disturbances in the metabolic activity of mitochondria, which has been shown to be associated with the ageing process and mtDNA depletion syndromes (MDS). This project will deliver key information about the role of DNA topoisomerase IIIalpha in mtDNA maintenance and its effect on the metabolic activity of mitochondria. With its deliverables it will open up new avenues for the benefit of the research community, for training and public engagement, end for exploitation of basic research findings in healthcare and commercial applications.

Our findings will be published in "open access" publications to ensure to a wide research community. Research findings will be communicated in international and national conferences before publication to receive helpful feedback and criticism. We will seek collaborative connections initially to share new and improved techniques ideas, preliminary data and reagents, then set up new collaborative projects in joint grant proposals.

It has been my ambition to maintain a laboratory portal for many years, and with the infrastructure provided by the University of Lincoln it will be set up maintained and updated for years to come. We will use this service to communicate our findings to expert scientists and to share "raw" experimental data, but also to communicate our findings to the wider audience to emphasise the importance of our research. In addition to public outreach through the website, we will maintain already existing links and set up new ones with Lincolnshire schools to promote interest in our work, and organise demonstrations and lectures to actively involve young people in science.

We are aiming to hire an experienced postdoctoral researcher, in return we will support her/his career development and aspirations to e.g. become an independent researcher or join a commercial enterprise in biotechnology. The PDRA will be actively involved in networking with academic and clinical researchers, and with potential industrial partners. We will recruit PhD and MSc students to complement research not only manually but with ideas as well, and to provide valuable technical and theoretical training.

We will seek collaborations with clinicians who provide treatment and care for mitochondrial DNA depletion syndrome sufferers, to set up collaborations for follow-up studies to test the involvement of Topo IIIalpha in the aetiology of these diseases. This will also open up avenues to identify "druggable" targets with potential commercial partners.

In our review process a high priority will be given to assess if the impact objectives are met and to identify new avenues of impact delivery. Person responsible for impact management will be the PI.
 
Description DNA topoisomerase IIIalpha (TOP3A) is a type 1A enzyme that is localised to the nucleus as well as the mitochondria, and maintains the topology of DNA in these organelles. Deletion or inactivating mutations are lethal in most organisms, and in cultured cells these cause cell death or senescence. In order to study the cellular function of this enzyme without the overall deleterious effect, we separated its nuclear and mitochondrial functions. We combined system-wide depletion of endogenous expression with re-expression of a variant that specifically localises to either the nucleus or the mitochondria.

We identified signals that under normal circumstances direct the protein to the nucleus or to the mitochondria. The amount of protein that goes to either of these organelles is also under tight controls. We identified the mechanisms how this regulation is achieved, and that it is a common mechanism used by a number of other proteins with similar dual localisation.

We confirmed phenotypes attributable to nuclear elimination of the protein, which have been seen by other laboratories in response to system-wide depletion. DNA damage was detected at the telomeres in cells that rely on homologous recombination for telomere maintenance (ALT), which was associated with telomere erosion and senescence. While mitochondrial expression was maintained in these cells, these findings confirm that development of senescence is due to perturbation of nuclear functions of TOP3A. We also found further signs of defects in maintenance of the nuclear genome. The frequency of sister-chromatid exchanges and micronuclei increased indicating major disturbances in chromosome maintenance, likely due to defects in processing homologous recombination intermediates.

We then used cells in which nuclear activity of TOP3A is maintained to avoid the effects of systemic depletion described above, but the enzyme is absent from the mitochondria. We measured mitochondrial activity in these cells and found that mitochondrial membrane potential was severely perturbed. We measured changes of mtDNA copy numbers and found that drop of mitochondrial activity was a consequence of mtDNA depletion. In a follow-up work we are analysing consequences of TOP3A depletion on the topological state of mtDNA.

These results indicate that TOP3A is indispensable for mtDNA maintenance, and mutations that only hinder its activity without complete inactivation might contribute to the mutation spectrum of mitochondrial diseases and mitochondrial DNA depletion syndromes.

We are currently characterising a likely nuclear interactor of TOP3A. We found that this hitherto uncharacterised protein interacts with proliferating nuclear antigen (PCNA), the small ubiquitin-like modifier SUMO2, and the Fanconi anaemia protein FANCI, and is involved in the processing of damaged DNA. Its depletion sensitises cells to genotoxic insults, induces persistent activation of the DNA damage response, and causes slow growth.
Exploitation Route Patients affected by mitochondrial diseases have been described in the literature, in which no associated mutations have been identified. Based in our findings we propose that hypomorphic alleles of TOP3A might exist and could contribute to mitochondrial diseases and mitochondrial DNA depletion syndromes despite the fact that inactivating mutations or complete depletion of TOP3A are lethal in most organisms. Furthermore, we propose that our novel uncharacterised interacting protein operates in the homologous recombination pathway. If it proves to be druggable it might serve as a target for cancer treatment either alone, or in synthetic lethal combinations with PARP inhibitors.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Training workshop in techniques to study protein-protein interactions with live cell imaging
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact The workshop was developed based on our expertise to study protein-protein interactions. The workshop is a standard element of our integrated Masters courses and optionally taken by postgraduate research students as well. The workshop builds on the basic level training in confocal microscopy the Institution provides to users and teaches advanced techniques, such as FRET, FRAP, PCA/BiFC.
 
Description School of Life Sciences Early Career Postgraduate Studentship
Amount £44,500 (GBP)
Organisation University of Lincoln 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description University of Lincoln College of Science Back to Science award
Amount £21,000 (GBP)
Funding ID 0000645 
Organisation University of Lincoln 
Sector Academic/University
Country United Kingdom
Start 11/2015 
End 10/2017
 
Description University of Lincoln Research Investment Fund
Amount £17,500 (GBP)
Funding ID A16970 
Organisation University of Lincoln 
Sector Academic/University
Country United Kingdom
Start 02/2014 
End 02/2015
 
Title Generation of inducible DR-GFP rescue cell lines 
Description This expression system combines the commercially available Tet-ON, Gateway and Flp-In (Life Technologies) technologies with the DR-GFP homologous recombination reporter in U2OS cell background. 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact siRNA technology is suffering from undesirable off-target side effects, for which a phenotype that is the result of siRNA knockdown should be verified using re-expression of the siRNA resistant target gene to rescue the phenotype. It is also possible to re-express mutated forms of the target gene to analyse the effect of mutations. The expression system we developed simplifies this verification and rescue process for the study of homologous recombination repair, generation of clonal rescue cell lines can be achieved in 3-4 weeks. 
 
Description Cellular uptake of ultra-small gold nanoparticles 
Organisation Midatech Pharma PLC
PI Contribution We are working on the characterisation of cellular uptake and effect of a proprietary gold nanoparticle design developed by Midatech. Midatech initiated the partnership based on expertise in my laboratory and my internal collaborator, Dr Enrico Ferrari.
Collaborator Contribution Midatech is funding an MSc by Research studentship in my laboratory. The student is jointly supervised by myself and Dr Enrico Ferrari in the School of Life Sciences, University of Lincoln.
Impact No outcome yet; the project is on-going. Multidisciplinary project involving chemical synthesis and biophysical methods.
Start Year 2018
 
Description Development of a nanodevice to deliver sequence-specific DNA damage 
Organisation University of Lincoln
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing a molecular and cellular testing platform for the nanodevice developed by our collaborator, who is an established organic chemist. The work is carried out by a part-time postdoctoral scientist; supported by the College of Science Back to Science award.
Collaborator Contribution Dr Ishwar Singh, our collaborating partner in the School of Pharmacy is contributing his the synthetic chemistry side of the project. They are synthesising the compounds we are testing.
Impact This is a multidisciplinary collaboration between an organic chemist and my laboratory. We applied to the College of Science back to Science program with this joint interdisciplinary project.
Start Year 2015
 
Description Development of antimicrobial surface coating for use on implants and prosthetics 
Organisation Q Technologies Ltd
Country United Kingdom 
Sector Private 
PI Contribution I am contributing a cellular platform for testing and development of new antimicrobial coating of implants and prosthetics.
Collaborator Contribution Quiesco technologies brings in the coating know-how and the antimicrobial compound.
Impact No outputs yet. This is a multidisciplinary collaboration that brings in the chemical development of coating compounds.
Start Year 2016
 
Description Functional characterisation of proteins involved in homologous recombination 
Organisation Zhejiang University
Country China 
Sector Academic/University 
PI Contribution We provided expertise in microscopic analysis and fluorescent in-situ hybridisation
Collaborator Contribution The research concept was devised by our partner, Dr Songmin Ying, and was developed jointly with the Helleday group in Stockholm, Sweden.
Impact doi: 10.18632/oncotarget.5497 doi: 10.2174/1574892808666131118232656
Start Year 2013
 
Description Generation of DR-GFP inducible expression system 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration partner in Oxford used the inducible expression system we developed for the current grant, expanded with the DR-GFP homologous recombination reporter. Mutant forms of KDM4A and SETD2 were expressed in an inducible manner following siRNA depletion of the respective endogenous gene products, and the effect of mutations of KDM4A and SETD2 on homologous recombination was directly studied via the DR-GFP reporter
Collaborator Contribution This is a multilateral collaboration and each partner contributed experiments, designs and ideas to the project and to the final publication output. Contribution of each author is listed in the publication output under 'Author Contributions'.
Impact DOI:10.1016/j.celrep.2014.05.026
Start Year 2013
 
Description Identification and characterisation of novel factors involved in homologous recombination repair 
Organisation Karolinska Institute
Country Sweden 
Sector Academic/University 
PI Contribution My postdoctoral fellow carried out parts of the screening and characterisation steps. We took on a number of hits from the screen for further functional characterisation, and to verify physical interaction with the Topoisomerase 1alpha-BLM-RMI1 complex.
Collaborator Contribution Major part of the large siRNA screen and characterisation was carried out in Professor Thomas Helleday's laboratory in the Science for Life Labratories at the Karolinska Institute
Impact DOI: 10.1038/celldisc.2015.34
Start Year 2013
 
Description Identification of interacting partners and post-translational modifications of TOP3A by mass spectrometry 
Organisation Nottingham Trent University
Department John van Geest Cancer Research Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We generated cell lines that express Emerald-GFP tagged TOP3A variants that are specifically targeted to either the nuclei or the mitochondria. We prepare samples by GFP-TRAP pulldown and separate co-purifying proteins on SDS polyacrylamide gels.
Collaborator Contribution Protein samples are extracted from the polyacrylamide gel slices we provide. Co-purifying protein species contained in the gel slices are identified by TripleTOF mass spectrometry and SWATH acquisition.
Impact Data acquisition is still ongoing, no output has been generated yet.
Start Year 2014
 
Description JIP2 and Tribbles-1 Interact to Regulate Vascular Smooth Muscle Cell Function 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution We have the infrastructure in our school for collecting microscopic images for quantitative analysis, and we developed expertise and experience to carry out the actual quantitative analysis. We are contributing this infrastructure and know-how to the collaboration.
Collaborator Contribution The collaborating partner is providing microscopic preparations for us to analyse.
Impact Adrienn Angyal, Hye Youn Sung, Csanad Bachrati and Endre Kiss-Toth (2015) Mitogen Activated Protein Kinase scaffold Jun Kinase-Interacting Protein 2 (JIP2) and Tribbles-1 Interact to Regulate Vascular Smooth Muscle Cell Function. Manusript under review.  
Start Year 2015
 
Description Size-dependent cellular uptake of exosomes 
Organisation University of Lincoln
Country United Kingdom 
Sector Academic/University 
PI Contribution My postdoctoral fellow contributed her extensive expertise in cell culturing and cellular assay techniques to the work with advice and and supervision and experimental design.
Collaborator Contribution This project introduced the concepts of exosomal communication between cells to our work. My postdoctoral fellow has developed project ideas of her own based on this collaboration and is currently seeking funding for further development of these ideas.
Impact doi: 10.1016/j.nano.2016.12.009
Start Year 2016
 
Description Unlocking the Potential of the Unexploited Antibiotics Moenomycin A and Teixobactin to Target Resistant Gram Negative Bacteria 
Organisation University of Lincoln
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing a human cell culture testing platform for the development of novel antibiotic conjugates that are developed by our organisc chemist collaborating partner.
Collaborator Contribution Dr Ishwar Singh in the School of Pharmacy is developing novel antibiotic conjugates. Dr Edward Taylor is contributing structural biology and microbiology assays to the project to confirm interaction of the novel conjugate with the target enzyme.
Impact This is a multidisciplinary project bringing together organic chemistry, structural biology, cell biology and microbiology. Patent applications: +PCT/GB2015/052564 UK patent application, 1415776.2, New Antibacterial Products doi: 10.1039/c7sc03241b
Start Year 2015
 
Description Unlocking the Potential of the Unexploited Antibiotics Moenomycin A and Teixobactin to Target Resistant Gram Negative Bacteria 
Organisation University of Lincoln
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing a human cell culture testing platform for the development of novel antibiotic conjugates that are developed by our organisc chemist collaborating partner.
Collaborator Contribution Dr Ishwar Singh in the School of Pharmacy is developing novel antibiotic conjugates. Dr Edward Taylor is contributing structural biology and microbiology assays to the project to confirm interaction of the novel conjugate with the target enzyme.
Impact This is a multidisciplinary project bringing together organic chemistry, structural biology, cell biology and microbiology. Patent applications: +PCT/GB2015/052564 UK patent application, 1415776.2, New Antibacterial Products doi: 10.1039/c7sc03241b
Start Year 2015
 
Title Improved quantification of co-localisation of proteins using CellProfiler 
Description Quantification of degree of co-localisation of proteins has several well established tools (Imaris CoLoc; InCell) however, they are all commercially available only. We developed a pipeline for the CellProfiler software platform that takes images automatically collected by a confocal microscope. Although the idea of this type of quantification is not new it is now improved and adapted to proteins that participate in the DNA damage response. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2015 
Impact The development is new, and has not resulted in impact or output yet. 
 
Description Applicant day presentations 
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 Schools
Results and Impact Prospective applicants to our undergraduate courses attend these Applicant Day presentations with friends and family. I am talking about the research we carry out in our laboratory. I concentrate on my interest in DNA metabolism, replication and the role of DNA topoisomerases, and how targeting DNA topoisomerases can lead to cancer treatment or the generation of new antibiotics.

This is a recurrent activity. When they eventually join us as undergraduates I often receive the feedback that my short presentation made a significant impact on their decision to come to us and possibly join to a research laboratory to do something similar.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
 
Description Cafe Scientific lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact "Café Scientifique is a place where, for the price of a cup of coffee or a glass of wine, anyone can come to explore the latest ideas in science and technology. Meetings have taken place in cafes, bars, restaurants and even theatres, but always outside a traditional academic context. Cafe Scientifique is a forum for debating science issues, not a shop window for science. We are committed to promoting public engagement with science and to making science accountable"

The relevance of my the talk was given by the decision of the Parliament to allow the generation of 'three parent babies' on the 3rd of February 2015. About 60-80 people attended my talk from various Schools of the University of Lincoln, and by members of the public. I concentrated on talking about the facts, and left the ethical debate and considerations to personal discussions afterwards. These discussions were engaging and interesting, often conflicting views were presented.
Year(s) Of Engagement Activity 2015
URL http://cafescilincoln.co.uk/2015/02/mom-dad-donor-and-the-mitochondrial-disease-by-dr-csanad-bachrat...
 
Description UoL - ULHT Joint research seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact The purpose of the meeting and presentations was to raise mutual awareness of biomedical research activities and interests conducted at both the University of Lincoln and at the United Lincolnshire Hospitals NHS Trust. I presented results and project plans related to my work on mitochondrial DNA maintenance and another area I am interested in, mesenchymal stem cell research.


My presentation attracted interest from clinicians, and I managed to identify potential collaborators in the Hospital. With one of the clinicians we are currently discussing a jointly funded PhD studentship.
Year(s) Of Engagement Activity 2013