Mechanics and execution of homologous recombination: a single-molecule view
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Author(s)
Belan, Ondrej
Type
Thesis
Abstract
Homologous recombination (HR) is an essential mechanism for the repair of toxic DNA double-strand breaks (DSBs), which, when not repaired accurately, can give rise to cancer and hereditary disorders. During HR, RAD51 forms helical nucleoprotein filaments on RPA-coated ssDNA with the help of mediator proteins (BRCA2 and RAD51 paralogs) and catalyses strand invasion into homologous duplex DNA. How this is achieved in not completely understood. To dissect the process on molecular level, I first reconstituted nematode RAD-51 presynaptic filament assembly in the presence of mediator proteins at the single-molecule level and demonstrated that BRC-2 promotes RAD-51 nucleation, while RAD-51 paralogs transiently bind 5’ RAD-51 filament ends to stimulate RAD-51 growth in a 3’ to 5’ direction. In the second part of the thesis, I investigated the consequences of a permanently ‘switching on’ RAD-51 by engineering a variant of human RAD51, I287T, that forms presynaptic complexes efficiently without the recombination mediators present and analysed its impact on cellular DNA metabolism. I showed that RAD51 I287T is toxic in cells as it interferes with genome duplication by promiscuously loading at replication forks. Lastly, I demonstrated that nematode RAD-51 is surprisingly tolerant to mismatches during DNA strand exchange catalysis. The mismatch tolerance can be abolished by engineering specific mutations into the DNA binding loop of RAD-51, which causes meiotic HR stalling in the absence of regulatory motor proteins. Together, this work has uncovered unappreciated mechanisms that promote and maintain optimal RAD51 filament assembly and how deviations to optimal assembly rates can lead to disease - a phenomenon referred to as the ‘Goldilocks principle’ of RAD51 assembly.
Version
Open Access
Date Issued
2021-09
Date Awarded
2022-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Boulton, Simon
Armada, David
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)