ESR12: “The role of RAD51 filament in genome stability and cancer”

ICRC (L. Krejčí group): The role of RAD51 filament in genome stability and cancer

i. Objective of research: Molecular characterization of RAD51 filament assembly and its impact on genetic disorders.

ii. Current state of the art: Homologous recombination (HR) is an essential mechanism for the repair of DNA double-strand breaks and damaged replication fork and is associated with genetic disorders, cancer and aging. HR repairs DNA damage by copying the genetic information from an intact chromosomal template, which is critically dependent on the RAD51 recombinase. To ensure its timely and accurate completion, HR is positively and negatively regulated by RAD51 co-factors and anti-recombinases. How these HR regulators function at the molecular level remains poorly understood and represents a significant challenge to the field due to the lack of mechanistic resolution afforded by conventional bulk biochemical approaches. The aim of our proposal is to extend this paradigm to study multiple different HR regulators to gain insights into how they work individually and how they act cooperatively during HR. Deciphering how HR regulators work will provide an improved understanding of the molecular mechanisms of carcinogenesis and may present unique opportunities for therapeutic intervention.

iii. Research methodology and approach: Our project is highly innovative as it will employ genetics, biochemistry and cutting-edge biophysical approaches, including high resolution Cryo-EM imaging, Switch-sense, stop-flow, and single molecule FRET assays to monitor the impact of HR regulators on key stages in the HR reaction. The real strength and synergy of our collaboration comes from the unique expertise of each team member covering different genetic, biophysical and structural methods. The aim of our proposal is to exploit this multi-disciplinary approach to unravel the molecular basis by which RAD51 regulatory factors act and cooperate during HR. These approaches in isolation have limited scope to inform on the precise mechanism of action of a particular regulatory factor but together they can inform on kinetics, affinities, protein dynamics and structural changes that arise during the HR reaction, as well as the biological implications of these mechanistic studies. Only by integrating the data from these different approaches will we be able to uncover the molecular basis of HR co-factor action, which will open up new research directions and will have a transformative impact on our field. Hence, our approach is critically dependent on collaboration and combining the respective expertise of the applicants. 

iv. Originality and innovative aspects of the ESR project: We will use recently pioneered biophysical and structural methods to monitor how RAD51 regulatory factors act and cooperate during HR .

v. Integration of the ESR project to the overall research programme: Our ESR will work with the Branzei group on genetic studies in yeast and biochemical studies in X. laevis egg extracts, with Norgenotech on identifying HR factors at telomeres and with the Polo and Legube groups on the role of HR factors on chromatin changes and its dynamics.