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Title: Mechanism of action of Rad51 paralogs
Author: Taylor, M. R. G.
ISNI:       0000 0004 5364 5558
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Homologous recombination (HR) is an essential DNA break repair mechanism that remains incompletely understood. HR is a complex multistep process initiated by the loading of RAD-51 recombinase as filaments onto single stranded DNA (ssDNA). This structure directly invades an intact homologous duplex, which serves as a template for repair DNA synthesis. Numerous positive regulators of HR have been described, including the Rad51 paralogs, but the mechanism of action of Rad51 paralogs in promoting HR is unknown. In this study, I have characterized the mechanism of action of a novel Rad51 paralog complex, RFS-1/RIP-1, from C. elegans. RFS-1 is a Rad51 paralog required for RAD-51 focus formation at stalled replication forks, indicating an early positive regulatory role in HR. I demonstrate that RFS-1 interacts with a nematode-specific orphan protein, RIP-1. I identify a cryptic Walker B ATPase-like motif within RIP-1, which is functionally important in establishing the RFS-1/RIP-1 interaction interface. rip-1 and rfs-1 mutant animals phenocopy for essentially all phenotypes analysed. Together these data suggest RFS-1/RIP-1 functions as a constitutive complex. I show recombinant RFS-1/RIP-1 can be purified and specifically binds ssDNA but lacks measurable ATPase activity. RFS-1/RIP-1 also strongly stimulates strand invasion activity by RAD-51, consistent with a pro-recombinogenic function in vivo. I define for the first time the mechanism of action underlying the intrinsic ability of Rad51 paralogs to stimulate HR. Using a combination of biochemical and biophysical approaches, notably electrophoretic mobility shift assays, stopped-flow reaction kinetics and nuclease protection assays, I show RFS-1/RIP-1 dramatically alters the properties of RAD-51-ssDNA filaments such that RAD-51 is more stably associated with ssDNA yet the ssDNA is more sensitive to nuclease degradation. RFS-1/RIP-1 exerts these effects primarily downstream of filament formation, ruling out a major role in RAD-51 loading. I propose RFS-1/RIP-1 remodels RAD-51-ssDNA filaments to a conformation poised for pairing with the template duplex and strand invasion.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available