Single-molecule visualisation of eukaryotic DNA replication termination to uncover novel mechanisms of replication stress

Project Information

SinMolTermination

Grant agreement ID: 794962

DOI

10.3030/794962

Project closed

EC signature date 28 March 2018

Start date 11 February 2019

End date 15 April 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 454,80

EU contribution

€ 183 454,80

Coordinated by

THE UNIVERSITY OF BIRMINGHAM
United Kingdom

Periodic Reporting for period 1 - SinMolTermination (Single-molecule visualisation of eukaryotic DNA replication termination to uncover novel mechanisms of replication stress)

Reporting period: 2019-02-11 to 2021-02-10

Summary of the context and overall objectives of the project

Perturbation to the replication of the human genome, so called replication stress, is a key endogenous source of genome instability that leads to ageing, neuropathology and cancer development. DNA replication occurs in three stages: initiation, elongation and termination. Fundamental knowledge about replication initiation and elongation led to the development of replication-stress inducing drugs that target rapidly dividing cancer cells. However, despite recent breakthroughs there is still little understanding about the molecular mechanism of termination and how it can generate replication stress. This is due to the recalcitrant nature of termination to traditional experimental approaches. We seek to fill this significant gap in knowledge within the eukaryotic DNA replication field by developing novel technology and approaches to study DNA replication termination. Our multidisciplinary approach will permit direct and continuous visualization of individual eukaryotic replication forks during replication elongation and termination. Our results are anticipated to provide a significant and unique breakthrough in the replication field, spearhead new avenues of cancer therapy development, as well as providing novel assays and research tools for the international research community.
We were successful in the creation of a single-molecule imaging approach to study replication termination but ultimately further progress and dissemination of results was prevented due to the Covid-19 pandemic.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The major aims of the project were to create a single-molecule assay with super-resolution capabilities that could be used to precisely track the movement of replication forks in Xenopus egg extract. We successfully created this technology and can visualize the movement of replication forks. Unfortunately, the Covid-19 pandemic hindered our progress and prevented us from completing the biological objectives of the fellowship, defining how replisomes terminate and how replication-stress can occur through mis-regulated termination. This work is still on-going and is expected to come to fruition after the fellowship period.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The Covid-19 pandemic completely hindered our ability to achieve the original scientific goals set out in the project proposal. That work is still on-going as we did manage to set up the key technology and infrastructure necessary for the completion of our scientific objectives. We anticipate that our results will be of high importance to the DNA replication and repair fields. The fellowship period stimulated novel discussions and collaborations, extending the scientific reach of the researchers and the impact they can have on their fields. A consequence of this has also been to re-think the use of animal-derived reagents (antibodies) and looking for alternative approaches which will have a positive effect on the reduction of animal usage in biomedical research. The interdisciplinary nature of the project has also meant exposure into new areas of microscopy and stimulated new lines of thinking to develop our technology even further than what we had originally imagined.

Proposed replication termination model and visualising DNA replication on single DNA molecules