Single molecule analysis of genome replication
Lead Research Organisation:
University of Oxford
Department Name: Sir William Dunn Sch of Pathology
Abstract
All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. Our research aims to determine how cells ensure that the replication of each chromosome is completed before cell division. Our experimental data have revealed the average pattern of chromosome replication in populations of millions of cells - that is the regions that are replicated first, second and so on. However, in preliminary data we have discovered that individual cells do not replicate their chromosomes with this 'average' pattern; the pattern of DNA replication in single cells is hidden by the population average. Consequently rare problems during DNA replication, including delays in completing replication, cannot currently be readily detected. Therefore, we are developing novel methods to directly measure DNA replication in single molecules. In one approach, we are using a transformative single-molecule sequencing technology to detect the pattern of DNA replication in thousands of long molecules. In addition, we will make use of recent advances in imaging technologies to directly visualise DNA replication in whole chromosomes. These complementary approaches will, for the first time, reveal the pattern of DNA replication on individual chromosomes. This is important because a single DNA replication error on one chromosome in a single cell division can give rise to genomic disorders, including cancer.
Technical Summary
Complete, accurate genome replication is essential for life. Our long-term goal is to determine how cells faithfully complete genome replication. Errors in DNA replication occur on single molecules in individual cells; however these errors are hidden from view in genomic approaches that look at data from populations of several million cells. Currently, there are no genomic methods to study DNA replication in single cells or molecules. We will close this gap by pioneering two independent single-molecule techniques to discover how the genomes of individual cells are replicated. First, we will exploit recent dramatic advances in single-molecule DNA sequencing to directly detect base analogues that were incorporated during a pulse-chase experiment. This builds upon our demonstration that the MinION nanopore sequencer can clearly distinguish between natural bases (such as thymidine) and a base analogue (such as bromodeoxyuridine, BrdU) on ultra-long DNA sequence reads (>100 kb). Second, we will make use of a high-resolution DNA visualisation platform (called Irys) to detect the pattern of DNA replication in chromosome-length molecules. This will allow us to determine the location of active replication origins and the location of replication termination sites, in thousands on individual molecules. Together these experiments will provide the first high-resolution, whole-genome view of chromosome replication in single molecules. Our work with both wild-type and perturbed cells will allow us to discover mechanisms that contribute to stable genome inheritance.
Planned Impact
Who will benefit from this research?
The proposed work has long-term healthcare implications that will be of potential benefit to a wide range of patient groups, in particular cancer sufferers. Disruption of the regulation of DNA replication contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Genomic regions with few active replication origins are hotspots for rearrangements in cancer. Outcomes from the proposed research will provide novel mechanistic insights to genome replication and the human diseases associated with its deregulation, including cancer and developmental disorders. Thus, the biomedical implications of this work fit within the BBSRC's Strategic Research Priority "Bioscience for health".
How will they benefit from this research?
This proposal aims to investigate chromosome replication in single molecules and determine the degree of cell-to-cell variability. We will work in the genetically tractable model system Saccharomyces cerevisiae. We anticipate that our results will be informative about genome replication in other eukaryotes, because the key proteins involved in binding and activating replication origins are conserved between yeast and humans. Furthermore, differences in replication time (and the features that they correlate with) are also found from yeast through to humans. Therefore, the results of this project could help determine why some genomic regions have a low abundance of active replication origins and this could be an essential step towards improved therapeutic intervention.
What will be done to ensure that they have the opportunity to benefit from this research?
In addition to traditional routes of publication, the outcomes from this project will be communicated through our web pages, the replication origin database (OriDB), the University of Oxford's Communications and Marketing Unit, Oxford's Café Scientifique, and the BBSRC media office. Potential future health benefits will be exploited via colleagues within the Medical Sciences Division.
Professional development for staff working on the project
The project offers many opportunities for the postdoctoral researchers to acquire additional skills. The collaborative nature of the research will expose both individuals to molecular biology, genomics, bioinformatics and mathematical modelling. The PDRAs will receive training and hands-on experience of advanced bioinformatics approaches for analysis of novel deep sequencing technologies. The scientific communication skills of the PDRAs will be fostered by presenting our research to academic audiences and the general public (e.g. Oxford's Café Scientifique or local schools). Appropriate training for both audiences will be provided by the University of Oxford Science Outreach Programme, at a Genetics Society Workshop on 'Communicating Your Science' and at a EMBO Laboratory Management Course for Postdocs.
The proposed work has long-term healthcare implications that will be of potential benefit to a wide range of patient groups, in particular cancer sufferers. Disruption of the regulation of DNA replication contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Genomic regions with few active replication origins are hotspots for rearrangements in cancer. Outcomes from the proposed research will provide novel mechanistic insights to genome replication and the human diseases associated with its deregulation, including cancer and developmental disorders. Thus, the biomedical implications of this work fit within the BBSRC's Strategic Research Priority "Bioscience for health".
How will they benefit from this research?
This proposal aims to investigate chromosome replication in single molecules and determine the degree of cell-to-cell variability. We will work in the genetically tractable model system Saccharomyces cerevisiae. We anticipate that our results will be informative about genome replication in other eukaryotes, because the key proteins involved in binding and activating replication origins are conserved between yeast and humans. Furthermore, differences in replication time (and the features that they correlate with) are also found from yeast through to humans. Therefore, the results of this project could help determine why some genomic regions have a low abundance of active replication origins and this could be an essential step towards improved therapeutic intervention.
What will be done to ensure that they have the opportunity to benefit from this research?
In addition to traditional routes of publication, the outcomes from this project will be communicated through our web pages, the replication origin database (OriDB), the University of Oxford's Communications and Marketing Unit, Oxford's Café Scientifique, and the BBSRC media office. Potential future health benefits will be exploited via colleagues within the Medical Sciences Division.
Professional development for staff working on the project
The project offers many opportunities for the postdoctoral researchers to acquire additional skills. The collaborative nature of the research will expose both individuals to molecular biology, genomics, bioinformatics and mathematical modelling. The PDRAs will receive training and hands-on experience of advanced bioinformatics approaches for analysis of novel deep sequencing technologies. The scientific communication skills of the PDRAs will be fostered by presenting our research to academic audiences and the general public (e.g. Oxford's Café Scientifique or local schools). Appropriate training for both audiences will be provided by the University of Oxford Science Outreach Programme, at a Genetics Society Workshop on 'Communicating Your Science' and at a EMBO Laboratory Management Course for Postdocs.
Publications
Aydogan M
(2019)
A free-running oscillator times and executes centriole biogenesis
Aydogan MG
(2020)
An Autonomous Oscillation Times and Executes Centriole Biogenesis.
in Cell
Batrakou DG
(2020)
DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family.
in Nature protocols
Boemo M
(2020)
The Beacon Calculus: A formal method for the flexible and concise modelling of biological systems
in PLOS Computational Biology
Boemo MA
(2019)
Mathematical modelling of a hypoxia-regulated oncolytic virus delivered by tumour-associated macrophages.
in Journal of theoretical biology
Description | Most genomic approaches to studying chromosome replication measure an ensemble population average from many millions of cells. Our previous work has demonstrated that the population average does not accurately represent DNA replication dynamics in individual molecules (de Moura et al., 2010). Therefore, single molecule approaches are essential to detect rare problems during DNA replication, including delays in completing replication, that cannot currently be readily detected and are likely to underlie many disease states. However, current single molecule methods for DNA replication analysis are low throughput, low resolution and generally do not reveal the genomic location. This has motivated us to establish a high-throughput genomics approaches to study DNA replication on ultra-long single molecules. DNA polymerase can incorporate base analogues into newly replicated DNA. Using nanopore DNA sequencing we have been able to directly identify the location of these base analogues. From the pattern of base analogue incorporation we have been able to infer replication initiation sites, termination sites, and positions where replication forks pause; all with single-molecule resolution. We have called this method D-NAscent (for detecting nucleotide analog signal currents on extremely long nanopore traces) and have validated it against established ensemble population average datasets. The resulting publication (Müller, C. A., Boemo, M. A. ... Nieduszynski, C. A. (2019). Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. Nature Methods, 16(5), 429-436.) and sharing of the associated software (https://dnascent.readthedocs.io/en/latest/) mark successful completion of our Milestones 1-4, 7 & 8). Work towards milestones 5 & 6 relate to an alternative single molecule approach, based around optical mapping. This approach will have lower spatial resolution, but should allow analysis on even longer molecules and at higher throughput than is currently possible by nanopore sequence of DNA. We have now collected a whole genome datasets by this approach and our initial analyses suggest that we will be able to determine replication dynamics on individual molecules of up to 1 Mb. Further analyses will be necessary before this work can be published. Our final milestone (9) involves applying our single molecule approaches to study genome replication in genome-stability mutants. Towards this goal we have collected data using D-NAscent from multiple mutants and are currently analysing the data. In summary, we have successfully developed two single molecule methods for the analysis of genome replication and fulfilled all of our initial milestones. There is wide adoption by the genome stability field of our approach and we and others are now actively applying this approach to a range of biological questions in various different model systems. |
Exploitation Route | The development of the D-NAscent approach to the study of genome stability has opened up a wide range of important biological questions that could not previously be addressed. One mark of recognition of the importance of our method is that it featured on the cover of the abstract book for the seminal conference in our field (the 2019 CSHL meeting on Eukaryotic DNA Replication & Genome Maintenance). We are already aware of multiple research groups from around the world that wish to apply this method within their own research. Furthermore, we are actively applying D-NAscent to the study of DNA replication within human, budding yeast and fission yeast cells; each with the objective of addressing fundamental biological questions regarding the regulation of genome stability. |
Sectors | Healthcare,Other |
URL | https://dnascent.readthedocs.io/en/latest/ |
Description | Significant impact within academia: The technology developed during this award (for single molecule analysis of DNA replication) has gained wide-spread adoption across the academic community. I anticipate adoption within non-academic research organisation, for example companies involved in biomedical research. |
First Year Of Impact | 2022 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | Role of Senataxins in resolving transcription-replication conflicts |
Amount | £387,880 (GBP) |
Funding ID | BB/W01520X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2022 |
End | 08/2025 |
Description | Single molecule detection of DNA replication errors |
Amount | £437,916 (GBP) |
Funding ID | BB/W006014/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2022 |
End | 06/2025 |
Title | D-NAscent |
Description | D-NAscent (detecting nucleotide analog signal currents on extremely long nanopore traces) is a technology to measure the incorporation of nucleotide analogues on ultra-long single DNA molecules. The published method includes cellular growth to incorporate nucleotide analogues in DNA, isolation of DNA followed by nanopore DNA sequencing, and a software package (DNAscent) to determine the location of nucleotide analogues within each sequencing read. The software uses a hidden Markov approach to differentiate between a nucleotide analogue and thymidine using the raw nanopore signal. In an experimental setup where the nucleotide analogue, BrdU, is incorporated into nascent DNA by replication forks, this software can be used to answer questions that were traditionally answered by DNA fibre analysis. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | The method is now being applied to a wide range of biological questions in a range of organisms by our research team and by many others around the world. |
URL | https://dnascent.readthedocs.io/en/latest/ |
Description | Computational/Mathematical Collaborations |
Organisation | Ontario Institute for Cancer Research (OICR) |
Country | Canada |
Sector | Academic/University |
PI Contribution | We performed the experiments and wrote the software mentioned in the project description. |
Collaborator Contribution | Our collaborators Professor Jared Simpson (Ontario Institute for Cancer Research) and Dr. Alessandro de Moura (University of Aberdeen Institute of Medical Sciences) provided computational guidance and advice throughout the project. Dr. de Moura visited our lab on two occasions to participate in lab retreats, and Professor Simpson visited our lab to offer guidance and advice on our approach. |
Impact | The outcome of these collaborations are the DNAscent software and single-molecule nanopore method mentioned in the project description. This was a multi-disciplinary project that encompassed biology, computer science, and mathematics. |
Start Year | 2016 |
Description | Computational/Mathematical Collaborations |
Organisation | University of Aberdeen |
Department | Institute of Medical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We performed the experiments and wrote the software mentioned in the project description. |
Collaborator Contribution | Our collaborators Professor Jared Simpson (Ontario Institute for Cancer Research) and Dr. Alessandro de Moura (University of Aberdeen Institute of Medical Sciences) provided computational guidance and advice throughout the project. Dr. de Moura visited our lab on two occasions to participate in lab retreats, and Professor Simpson visited our lab to offer guidance and advice on our approach. |
Impact | The outcome of these collaborations are the DNAscent software and single-molecule nanopore method mentioned in the project description. This was a multi-disciplinary project that encompassed biology, computer science, and mathematics. |
Start Year | 2016 |
Description | Earlham-Aberdeen collaboration |
Organisation | University of Aberdeen |
Department | School of Medical Sciences Aberdeen |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have trained a post-doc from the collaborators research group in experimental and computation technologies for single-molecule analysis of DNA replication. |
Collaborator Contribution | The collaborators group have contributed technical expertise in the role of the Rif1 protein in regulation of DNA replication. |
Impact | Ongoing collaboration. |
Start Year | 2022 |
Description | Earlham-Cardoso lab collaboration |
Organisation | Technical University of Darmstadt |
Country | Germany |
Sector | Academic/University |
PI Contribution | Sharing of experimental and computational technologies for single molecule analysis of DNA replication |
Collaborator Contribution | Sharing expertise in mouse DNA replication control. |
Impact | Ongoing collaboration. |
Start Year | 2022 |
Description | Earlham-GDSC collaboration |
Organisation | University of Sussex |
Department | School of Life Sciences Sussex |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We are contributing single-molecule expertise in DNA replication to our joint collaborative project to determine the role of Senataxins in protecting genome stability. This work is funded by a joint BBSRC grant. |
Collaborator Contribution | The collaborators bring expertise in genome stability research and preliminary data on the role that Senataxins may play. |
Impact | Ongoing collaboration. |
Start Year | 2022 |
Title | DNAscent |
Description | DNAscent is software designed to detect the modified base BrdU in Oxford Nanopore reads. It uses a hidden Markov approach to differentiate between BrdU and thymidine using the raw nanopore signal. In an experimental setup where BrdU is incorporated into nascent DNA by replication forks, this software can be used to answer questions that were traditionally answered by DNA fibre analysis. |
Type Of Technology | Software |
Year Produced | 2019 |
Open Source License? | Yes |
Impact | The DNAscent software is being widely adopted by the chromosome biology and genome stability fields as evidenced by multiple requests to collaborate and many downloads of the software. |
URL | https://dnascent.readthedocs.io/en/latest/ |
Title | bcs |
Description | This software simulates models written in the Beacon Calculus, a process algebra for biology developed with this award. |
Type Of Technology | Software |
Year Produced | 2019 |
Open Source License? | Yes |
Impact | The software (and corresponding language) was designed to make modelling biological systems fast and easy (e.g., whole-genome DNA replication in budding yeast can be modelled in six lines of code). The corresponding manuscript is currently in press at PLoS Computational Biology as a methods paper. |
URL | https://beacon-calculus-simulator.readthedocs.io/en/latest/ |
Description | Catching the Science Bug: STEM Apprentice Placements Programme |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Two high achieving A-level students from Oxfordshire were hosted in our laboratory for one week. The students worked alongside experienced researchers to gain valuable insight into the work of a molecular biologist. Specifically, the students learned to culture and genotype yeast cells. Both students expressed an increased passion for science and the aim "to work hard and become a scientist. |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.path.ox.ac.uk/news/catching-science-bug-apprentice-placement-scheme-success-dunn-school |
Description | Cheltenham Science festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | A stand at Cheltenham Science festival to demonstrate the fundamentals of DNA (transcription, translation, replication) to families, using our own custom made dynamic DNA resources. |
Year(s) Of Engagement Activity | 2017 |
Description | Cherwell School (Oxford) - seminar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Seminar to local sixth-formers (Cherwell School, Oxford): "Nature versus nurture: the story of your genome" |
Year(s) Of Engagement Activity | 2017 |
Description | Hosting STEM students |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Two STEM students were hosted in the lab. They did experiments under supervision and attended seminars. |
Year(s) Of Engagement Activity | 2017 |
Description | Oxford UNIQ summer school |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Oxford UNIQ summer school. Members of the research group demonstrated protein purification techniques to a group of ~30 pre-university students. Aim to encourage an interest in science and to increase access to university/OxBridge. |
Year(s) Of Engagement Activity | 2017 |
Description | Phenotype (Oxford Biochemistry) magazine |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Undergraduate students |
Results and Impact | Phenotype (Oxford Biochemistry) magazine : a member of the research group wrote an written on science activism, interviewed the Oxford Climate Society, and performed copy editing for other articles. |
Year(s) Of Engagement Activity | 2017 |
URL | https://issuu.com/phenotypejournal/docs/mt17-1 |
Description | Phenotype (Oxford Biochemistry) magazine article |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Undergraduate students |
Results and Impact | Phenotype Oxford Biochemistry magazine: article "How to replication a genome" |
Year(s) Of Engagement Activity | 2018 |
URL | https://issuu.com/phenotypejournal/docs/phenotype_ht18_final_47c273f1b0a4d8 |
Description | School visit (Wolvercote primary school, Oxford) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Two year 6 science week session of 1.5 hrs were delivered in Wolvercote primary school using custom (designed and made) dynamic DNA resources to introduce pupils to genetic information, DNA structure including base pairing, and how it is replicated. Both the school (teachers) and pupils (feedback forms) enjoyed the activities and reported increased interest in the subject area. |
Year(s) Of Engagement Activity | 2017 |
Description | Science in schools: bringing antibiotics, bacteria and DNA to Oxfordshire students |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | A series of workshops have been created, centred on two themes: antibiotic resistance ('The Antibiotics Crisis') and DNA ('Dynamic DNA'). 'The Antibiotics Crisis' was developed with Science Oxford, and instigated and overseen by the Museum of the History of Science to celebrate 75 years (2016) since the first human penicillin trials, complementing their 'Back from the Dead' exhibit. Students in Key Stages 3-5 can learn about bacteria and viruses, common diseases associated with them, and the antibiotics used to treat these illnesses. Further, students are introduced to the concept of antibiotic resistance and how antibiotics are discovered in modern science. 'Dynamic DNA' consists of hands-on activities predominately for Key Stage 2 students, bridging a syllabus gap prior to secondary school, where genetics begins to be introduced. Novel resources, such as 3D DNA nucleotides, have been created to help teach a complex topic in a fun and simple manner. Additionally, the 3D pieces are structurally correct, and could be used to teach more complex topics such as translation and transcription to A-level students. These workshops have been trialled in schools or with children of lab members, and will be delivered to schools in Oxfordshire in the next few years. |
Year(s) Of Engagement Activity | 2016,2017 |