Mäeots, Märt-Erik;
(2023)
Development of time-resolved cryo-EM and studying the mechanism of homology search by the RecA recombinase.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Structural determination and analysis have become increasingly prominent in recent years. At the forefront of this development has been the progress of cryo-electron microscopy (cryoEM). This technique involves the freezing of samples in cryogenic liquid to create vitrified ice, which has the property of being compatible with biological function. Due to this property, cryoEM can trap reaction intermediates. In principle it is possible with cryo-EM to freeze any conformation that a protein may adopt, but in practice most samples are frozen at equilibrium, due to the manual preparation of the sample. In this thesis I implement a new sample preparation method based on microfluidics and gas-assisted spraying. This new method is validated to show it achieves millisecond scale time-resolution by measuring the growth of RecA filaments and demonstrates ability to reach near-atomic resolution. DNA damage repair (DDR) is a collection of different pathways that respond to both endogenous and exogenous DNA lesions and maintain genome integrity. One of the most deleterious lesions is a double-strand break (DSB). Homologous recombination (HR) is an error-free pathway for the repair of these. In this process DNA is bound by a class of proteins known as recombinases, which function to facilitate the search for a homologous stretch of DNA in the rest of the genome. However, the mechanistic basis for this homology search remains unclear. Here I apply the newly developed method of time-resolved cryo-EM (trEM) to trap early intermediates of the homology search process. I present time-resolved, high-resolution structures of RecA showing the action of loop 1 in recognising the differences between the different DNA binding states. The structure also reveals global conformational rearrangement of the filament groove. The structures reveal the preferential binding locations for dsDNA during homology search and demonstrate the application of time-resolved sample preparation to address novel biological questions.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of time-resolved cryo-EM and studying the mechanism of homology search by the RecA recombinase |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10177485 |
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