Structure-function studies of the tumour suppressor complex RAD51B-RAD51C-RAD51D-XRCC2
File(s)
Author(s)
Greenhough, Luke
Type
Thesis
Abstract
Homologous recombination is critical for the protection and restart of stalled replication forks and the repair of chromosome breaks. Individuals with mutations in key homologous recombination genes including BRCA2, PALB2 and the RAD51 paralogs are predisposed to breast and ovarian cancer, and the cancer-predisposing syndrome Fanconi anaemia. BRCA2 is a molecular chaperone that orchestrates the assembly of RAD51-ssDNA helical nucleoprotein filaments, which subsequently drive the pairing of damaged DNA with the homologous sister chromatid. In humans, there are five RAD51 paralog proteins which assemble into two heterotypic protein complexes: BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) and CX3 (RAD51C-XRCC3). RAD51 paralog gene knockouts cause embryonic lethality, whereas cells deficient for the RAD51 paralogs display growth defects, reduced RAD51 focus formation, sensitivity to PARP inhibitors and replication fork defects. While BCDX2 and CX3 both contribute significantly to genome maintenance, their molecular functions remain unknown as biochemical and structural studies have lagged behind genetic and cellular studies.
Here, we have purified soluble BCDX2 and solved its structure at 2.2 Å resolution using cryo-electron microscopy. Remarkably, the arrangement of RAD51C-RAD51D-XRCC2 is reminiscent of three RAD51 protomers in a filament, whereas RAD51B-CTD is dynamic. Biochemical and single-molecule analyses reveal that BCDX2 stimulates the assembly of RAD51 nucleoprotein filaments in reactions dependent on the coupled ATPase activities of RAD51B and RAD51C. Critically, ATP hydrolysis by RAD51C induces a transient interaction with the mobile RAD51B-CTD, which in turn remodels and primes the RAD51CL1 loop for high-affinity ssDNA binding and subsequent RAD51 filament assembly. In summary, these studies provide critical contributions to the field of homologous recombination by providing the first insights into the structure-function relationship of the enigmatic BCDX2 tumour suppressor complex.
Here, we have purified soluble BCDX2 and solved its structure at 2.2 Å resolution using cryo-electron microscopy. Remarkably, the arrangement of RAD51C-RAD51D-XRCC2 is reminiscent of three RAD51 protomers in a filament, whereas RAD51B-CTD is dynamic. Biochemical and single-molecule analyses reveal that BCDX2 stimulates the assembly of RAD51 nucleoprotein filaments in reactions dependent on the coupled ATPase activities of RAD51B and RAD51C. Critically, ATP hydrolysis by RAD51C induces a transient interaction with the mobile RAD51B-CTD, which in turn remodels and primes the RAD51CL1 loop for high-affinity ssDNA binding and subsequent RAD51 filament assembly. In summary, these studies provide critical contributions to the field of homologous recombination by providing the first insights into the structure-function relationship of the enigmatic BCDX2 tumour suppressor complex.
Version
Open Access
Date Issued
2023-03-24
Date Awarded
01/10/2023
License URL
Advisor
West, Stephen
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)