Structural and biochemical studies of RAD51 filament modulators
File(s)
Author(s)
Carver, Alexander
Type
Thesis
Abstract
RAD51 filament formation and disassembly is essential for the faithful repair of double strand breaks and the protection and restart of stalled replication forks. Many proteins are involved in the regulation of RAD51 filament dynamics including BRCA2 and RAD54. This thesis presents a biochemical, biophysical and structural examination of the RAD51 filament disassembler Fidgetin-like 1 (FIGNL1).
FIGNL1 is an AAA+ protein that has been implicated in ciliogenesis, axonal transport and chromosome segregation through its regulation of microtubule dynamics. A number of recent studies have elucidated its role, in the repair of DNA and resolution of replication stress, as a RAD51 filament disassembling protein. However, how FIGNL1 disassembles RAD51 at a molecular level and the mechanism of activity is not known.
To dissect how FIGNL1 functions, we first reconstituted RAD51 filament disassembly using biochemical and biophysical methods. We revealed that FIGNL1 is able to disassemble RAD51 filaments on both single-strand and double-strand DNA substrates. Using mutagenesis, we determined that the ATPase activities of FIGNL1 and RAD51, and the RAD51-binding domain of FIGNL1, are essential for filament disassembly.
Next, using cryogenic electron microscopy, we showed that wild-type FIGNL1 forms a planar hexamer in the presence of nucleotide. In the presence of RAD51, we were able to resolve a 3.2Å cryo-EM reconstruction of the FIGNL1 hexamer in a split washer configuration with a peptide of RAD51 coordinated in the central hexameric pore. Using biochemical approaches, we showed that mutation of pore loop 1 of FIGNL1 results in loss of filament disassembly, and deletion of the RAD51 N-terminus inhibits filament disassembly by FIGNL1...
FIGNL1 is an AAA+ protein that has been implicated in ciliogenesis, axonal transport and chromosome segregation through its regulation of microtubule dynamics. A number of recent studies have elucidated its role, in the repair of DNA and resolution of replication stress, as a RAD51 filament disassembling protein. However, how FIGNL1 disassembles RAD51 at a molecular level and the mechanism of activity is not known.
To dissect how FIGNL1 functions, we first reconstituted RAD51 filament disassembly using biochemical and biophysical methods. We revealed that FIGNL1 is able to disassemble RAD51 filaments on both single-strand and double-strand DNA substrates. Using mutagenesis, we determined that the ATPase activities of FIGNL1 and RAD51, and the RAD51-binding domain of FIGNL1, are essential for filament disassembly.
Next, using cryogenic electron microscopy, we showed that wild-type FIGNL1 forms a planar hexamer in the presence of nucleotide. In the presence of RAD51, we were able to resolve a 3.2Å cryo-EM reconstruction of the FIGNL1 hexamer in a split washer configuration with a peptide of RAD51 coordinated in the central hexameric pore. Using biochemical approaches, we showed that mutation of pore loop 1 of FIGNL1 results in loss of filament disassembly, and deletion of the RAD51 N-terminus inhibits filament disassembly by FIGNL1...
Version
Open Access
Date Issued
2023-12-07
Date Awarded
2024-05-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Zhang, Xiaodong
Sponsor
Breast Cancer Now
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)