Mechanisms Regulating Genome Replication

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

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. Eukaryotes, such as yeast and people, have large genomes with millions of bases encoding the genetic information. To ensure complete replication of these genomes within the allowed time, the process of DNA replication starts at multiple sites along each chromosome, called replication origins. These replication origins are specialised DNA sequences that assemble the cellular machinery that then moves along the DNA, reading and copying the genetic material. It is essential that the cell activates sufficient replication origins to ensure complete replication of the chromosomes. The importance of controlling replication origin activation is highlighted by the genome instability and diseases that may result from uncontrolled chromosome replication. Despite the importance of DNA replication origins we understand little about the DNA sequences that specify and control them. Failures in the processes of DNA replication lead to genetic instability and diseases such as cancer and congenital disorders. In the future, a better understanding of the basic biology that ensures genetic integrity will give new insights that will allow improved diagnosis and treatment of these diseases.

We aim to understand how replication origin activation time is controlled. To study this process we have compared genome replication in different organisms. Primarily we work with baker's yeast, since this is safe, cheap and ethical, but most importantly all the steps of genome replication are similar between baker's yeast and people. Therefore advances we make working with yeasts will be informative for future studies and treatment of people. Recently we have found that the patterns of genome replication are very similar in different yeast species (in evolutionary terms equivalent to comparing people with birds). These comparisons allowed us to discover individual replication origins that show dramatic differences in activity between the yeast species. Now we will investigate what is responsible for this difference in origin activation time between the two species. Our experiments suggest that DNA 'regulatory' sequences close to the origin are responsible. Now we want to find these sequences and determine how they alter the activation time of the replication origin.

The similarities between genome replication in yeast and people mean that the discovery of these regulatory sequences in yeast may be informative about how replication origin activation time is regulated in people. If too few replication origins activate, parts of the genome will fail to replicate and this can result in cancer and developmental diseases. Therefore, in the future, a better understanding of how replication origin activation time is regulated may allow the development of improved treatments for these diseases.

Technical Summary

Complete, accurate genome replication is essential for life. In yeast and metazoans, regions of the genome replicate at a characteristic time during S phase and this is determined by the time at which replication origins activate. Replication timing correlates with, and may regulate or be regulated by, transcription levels, chromatin state, sub-nuclear positioning, stem-cell reprogramming and cellular differentiation. Furthermore, disruption of replication timing in cancer cells contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Thus, understanding the regulation of replication timing, specifically the mechanisms that determine when origins activate, will help elucidate the cell-cycle defects that lead to cancer.

We aim to determine the mechanisms that regulate replication origin activation time, by comparing origin activity in a range of species. We will use budding yeasts because of the conservation of genome replication with metazoans and the powerful genetic and comparative genomic approaches available. This proposal builds upon our observation that genome replication in different yeast species is highly conserved, but that individual origins differ in activation time between species or strains. We have discovered that these differences in activity arise as a result of cis-acting sequences. Therefore, these origins are particularly significant because they offer an opportunity to discover the mechanisms that regulate origin activation time. We now want to isolate these sequences and characterise the molecular mechanisms responsible for differences in origin activity. Understanding these mechanisms will be crucial for determining why regions of the genome replicate at characteristic times during S phase and how mistakes can give rise to diseases including cancer.

Planned Impact

Who will benefit from this research?

The long-term healthcare implications of this work are of potential benefit to a wide range of patient groups, particularly cancer sufferers. Our project aims to understand how genome replication time is regulated by the timely activation of replication origins. Disruption to replication timing contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Genomic sites with a low abundance of active origins have been found to be fragile sites and hotspots for rearrangements in various cancers. Therefore, the biomedical implications of this work respond to the BBSRC's Strategic Research Priority 3 ("Basic bioscience underpinning health").

How will they benefit from this research?

This proposal aims to identify the molecular mechanisms that regulate replication origin activation time. We will work in the genetically tractable model system Saccharomyces cerevisiae.
We anticipate that our results will be informative about the regulation of origin activation time 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 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 the traditional routes of publication, the outcomes from this project will be communicated to target audiences through our own web pages, the replication origin database (OriDB), the University of Nottingham's Communications unit, Nottingham's Café Scientifique, Nottingham's BioCity and the BBSRC media office. The potential future health benefits will beexploited primarily through opportunities to communicate with colleagues within the Faculty of Medicine and Health Sciences, in particular those in the Division of Pre-Clinical Oncology.

Professional development for staff working on the project

This project will offer opportunities for Ms Müller to acquire additional skill sets. These will include training in quantitative trait analysis and the informatics skills required to analyse the deep sequencing data generated. Opportunities to further develop communications skills will be provided both at scientific conferences, within the University of Nottingham, but also to non-specialist audiences through our work with the general public including schools.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/K007211/1 01/08/2013 31/08/2014 £346,733
BB/K007211/2 Transfer BB/K007211/1 01/09/2014 31/07/2016 £230,971
 
Description So far we have discovered SNPs that abolish replication origin activity (Milestone 1) and origin proximal polymorphisms that regulate origin activity in cis (Milestone 2). Our detailed characterisation of candidate origins (Milestone 4) has allowed us to generate functional origin chimaeras and discover the cis-acting sequences responsible for differences in origin activity (Milestone 5). The final stage of the project involved characterising the sequence elements that we discovered to uncover the molecular mechanism that regulate replication origin activity. First, we tested whether the elements we have identified protect the ORC-binding site from pervasive transcription. Our results clearly indicate that transcriptional differences are not responsible for the differences in origin behaviour. Second, we have shown that differences in the ORC-binding site are not responsible for the differences in origin behaviour. Although we are yet to positively assign function to the cis-acting sequences that we have discovered, we have been able to rule out two potential mechanisms. Further experiments will be required to conclusively determine how the cis-acting sequences regulate replication origin activity.
In parallel experiments we have compared the temporal control of DNA replication in a wide range of yeast species. These data allowed us to identify genetic elements with evolutionary conserved DNA replication times. From this we have been able to demonstrate that DNA replication time is a novel regulator of gene expression, specifically for cell cycle regulated genes.
Exploitation Route The mechanisms that regulate genome replication that we are identifying will be important to further our understanding of genome stability, particularly how completion of genome replication is regulated.
Sectors Other

 
Description Wellcome Trust Investigator Award
Amount £1,279,523 (GBP)
Funding ID 110064/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 05/2021
 
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 Schools outreach 
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 Presentation about 'Genome Science' to science pupils at Tunbridge School. The presentation included an opportunity for pupils to test there own response to a taste test and then understand the pattern of inheritance. This generated lively discussions and many questions. I had positive feedback from the schools, who emphasised the value to pupils deciding on university-level courses.
Year(s) Of Engagement Activity 2013,2014
 
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