Title: Time-resolved structural studies by cryo-electron microscopy
Funder: The Francis Crick Institute

Abstract: Molecular biology research aims at obtaining a comprehensive mechanistic description of cellular physiology. In general, the best-case outcome of most biochemical studies is the structural and kinetic characterization of their underlying chemical processes. Since no single experimental technique can directly deliver such information in a broadly applicable manner, structural studies of usually static macromolecules are complemented by kinetic characterization and molecular dynamics simulations. In practice progress is often impeded by the typical requirement for large amounts of labelled or crystalized samples. Moreover, the widely-used strategy of utilizing mutants or crosslinking probes to stabilize otherwise short-lived catalytic intermediates may be prone to artefacts and precludes time-resolved tracking of molecular events. My group is developing a method that largely overcomes these limitations by allowing the direct observation of biochemical processes at atomic spatial- and milliseconds time-resolution by combining microfluidics and cryo electron microscopy (cryo-EM). We utilize microfluidic devices to mix and incubate biochemical samples on sub-second time scales and then rapidly spray-plunge-freezes the reactants for subsequent three-dimensional structure determination by cryo-EM and single particle analysis. Iterating the procedure at increasing incubation times after sample mixing, allows the visualization of a biochemical binding and/or enzymatic reaction as a time-lapse “movie”. The core biological interest of the group are two-fold: elucidating the structural basis for the molecular mechanisms of DNA double strand break repair by homologous recombination and the regulation of ubiquitin signaling. Genome integrity is essential for organismal health and reproduction. However, the chemical environment and cellular metabolism frequently cause DNA damage, counteracted by multiple repair pathways. Intriguingly, homologous recombination repair (HRR) is solely responsible for the nearly error-free restoration of DNA double-strand breaks and is of tremendous importance to human health and gene technology and therapy. Although many HRR components are amenable to in vitro analysis, their functionally relevant interactions are often transient and recalcitrant to conventional trapping techniques. This limits the insights of structural studies and impedes the development of better-targeted diagnostics and therapy. The key questions we are pursuing revolve around uncovering the structural and kinetic basis for specificity during Rad51-mediated homology search. Following commitment to HRR, Rad51 filaments form on resected DNA, and invade intact chromatin in a trial-and-error search of a homologous sequence to serve as template for subsequent repair. Vexingly, this process requires both high speed, to sample the genome’s large search space, and high specificity, to ensure error-free repair. The balance is achieved through rapid sampling of invaded DNA by multiple, low-affinity, transient interactions. We will initially visualize such microhomology search intermediates of the bacterial recombinase RecA by time-resolved cryo-EM analysis to elucidate structural specificity determinants and directly correlate them to kinetic behaviour. We will extend these studies to human Rad51 and paralogues, and comprehensively describe the defining biochemical step of homologous recombination.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.4 METHODOLOGIES AND MEASUREMENTS
Health categories: GENERIC HEALTH RELEVANCE