Structural analysis of Rad51 paralogues involved in recombinational DNA repair

Project Information

SARPBAXZ

Grant agreement ID: 101022335

DOI

10.3030/101022335

Project closed

EC signature date 18 March 2021

Start date 1 May 2021

End date 30 April 2023

Funded under

• SOCIETAL CHALLENGES - Europe In A Changing World - Inclusive, Innovative And Reflective Societies

Total cost

€ 224 933,76

EU contribution

€ 224 933,76

Coordinated by

IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE
United Kingdom

Periodic Reporting for period 1 - SARPBAXZ (Structural analysis of Rad51 paralogues involved in recombinational DNA repair)

Reporting period: 2021-05-01 to 2023-04-30

Summary of the context and overall objectives of the project

Cancer formation is driven by genomic instability, which is caused by defects in DNA repair. A major DNA repair pathway is homologous recombination (HR). The Rad51 protein is central to HR, but numerous additional factors collaborate with Rad51. These regulatory proteins are varied in function. While there is significant structural information available for most of these auxiliary factors, one group known as the Rad51 paralogs is poorly studied due to their biochemical intractability. Furthermore, the interplay between these different auxiliary factors and their regulation at the post-translational level remains largely unexplored. Here, we examined these under-appreciated phenomena, with the aim of improving our understanding of HR. HR is not only integral to maintaining genome stability, but it is also involved in several other aspects of chromosome biology, such as DNA replication, meiosis, and telomere maintenance. Thus, the findings of this work have the potential to impact upon a wide variety of disciplines within biology. The technical challenges of structural studies with these proteins proved too much for us to overcome in the timeframe of the action. However, we were able to uncover new insights into how the Rad51 paralogs function with other auxiliary factors and how post-translational modifications may regulate the activation of Rad51 by these factors.

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

Significant time was spent getting to grips with the fundamentals of electron microscopy. Several attempts were made to visualise different auxiliary factors — the Rad51 paralogs Rad55-Rad57, Swi5-Sfr1, and Rad54 — bound to Rad51 filaments, but these attempts ultimately proved unsuccessful. In parallel, biochemical experiments were devised to investigate whether phosphorylation modulates the interaction of Swi5-Sfr1 with Rad51. This line of enquiry proved fruitful and we discovered that phosphorylation of Sfr1 negatively regulates the binding to and activation of Rad51, leading to less efficient DNA repair. These results were disseminated in several ways. Firstly, they were presented locally to other lab members in lab meetings. Secondly, I conducted this project with a MSc student that I was responsible for supervising, and this student also presented the project locally to obtain his masters degree. Thirdly, this project was presented at departmental meetings and symposia, both at Imperial College London and at the Francis Crick Institute, where we are currently on secondment. Fourthly, this work has just been accepted for publication in the Journal of Biological Chemistry, an international peer-reviewed scientific journal. There has not yet been an opportunity to disseminate these findings to the general public but efforts will be made going forward to utilise any opportunities that do arise.

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 work that was conducted here is discovery science, therefore there is no immediate impact on society. However, given that the research is of a biomedical nature, it has uncovered new insights into the mechanisms of DNA repair that may potentially be informative in better understanding human diseases such as cancer and conditions such as fertility.

Summary of how phosphorylation of Swi5-Sfr1 regulates Rad51-dependent DNA repair