Different kinds of lesions can occur to DNA, and among them, one of the most dangerous is the double strand break (DSB). DSBs can result in mutations, chromosome translocation or deletion. For this kind of lesions, depending on cell cycle phase as well as DNA-end resection, cells have developed specific repair pathways. Among these the error-free homologous recombination (HR) plays a crucial role. HR takes place during S/G2 phases, since the sister chromatids can be used as homologous templates. In this process, hRAD51 and BRCA2 are key players. hRAD51 is a recombinase of 339 amino-acids highly conserved through evolution that displays an intrinsic tendency to form oligomeric structures. BRCA2 is a very large protein of 3418 amino-acids, essential for the recruitment and accumulation of hRAD51 in the nucleus repairing-foci. BRCA2 interacts with hRAD51 through eight, so-called, BRC repeats, composed of 35-40 amino acids. Mutations within this region have been linked to an increased risk of ovarian cancer development. Several reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with hRAD51. To date the only interaction that has been structurally elucidated, is the one taking place amid the fourth BRC repeat and hRAD51. Nevertheless, due to the structural complexity and dynamics of RAD51, the mechanistic details of each step of RAD51 recruitment and DNA repair remain elusive. To shed light on the mechanism of hRAD51 defibrillation driven by BRC4, in presence or absence of co-factors, negative staining transmission electron microscopy experiments were combined with size exclusion chromatography data revealing that BRC4 erodes hRAD51 fibrils from their termini and does not attack the fibril at random positions. Nevertheless, the propensity to oligomerization of the WT protein hampered further biophysical studies. A novel stabilized fully human monomeric hRAD51 allowed us to investigate its interaction with BRC4 through orthogonal biophysical methods. SAXS experiments were also carried out on the hRAD51-BRC4 complex to provide novel structural insights on their behavior in complex. Atomistic modelling of generated Alphafold2 models revealed that both proteins display flexible N-terminal domains. These results, along with previous evidence on hRAD51 WT fibrils, suggest that BRC4 binding triggers a conformational rearrangement on the hRAD51 N-terminal domain from a more ordered to an intrinsically disordered state.

Rinaldi Francesco, G.S. (2023). Elucidating the BRCA2-RAD51 interaction through an integrated structural biophysics approach.

Elucidating the BRCA2-RAD51 interaction through an integrated structural biophysics approach

Rinaldi Francesco;Cavalli Andrea
2023

Abstract

Different kinds of lesions can occur to DNA, and among them, one of the most dangerous is the double strand break (DSB). DSBs can result in mutations, chromosome translocation or deletion. For this kind of lesions, depending on cell cycle phase as well as DNA-end resection, cells have developed specific repair pathways. Among these the error-free homologous recombination (HR) plays a crucial role. HR takes place during S/G2 phases, since the sister chromatids can be used as homologous templates. In this process, hRAD51 and BRCA2 are key players. hRAD51 is a recombinase of 339 amino-acids highly conserved through evolution that displays an intrinsic tendency to form oligomeric structures. BRCA2 is a very large protein of 3418 amino-acids, essential for the recruitment and accumulation of hRAD51 in the nucleus repairing-foci. BRCA2 interacts with hRAD51 through eight, so-called, BRC repeats, composed of 35-40 amino acids. Mutations within this region have been linked to an increased risk of ovarian cancer development. Several reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with hRAD51. To date the only interaction that has been structurally elucidated, is the one taking place amid the fourth BRC repeat and hRAD51. Nevertheless, due to the structural complexity and dynamics of RAD51, the mechanistic details of each step of RAD51 recruitment and DNA repair remain elusive. To shed light on the mechanism of hRAD51 defibrillation driven by BRC4, in presence or absence of co-factors, negative staining transmission electron microscopy experiments were combined with size exclusion chromatography data revealing that BRC4 erodes hRAD51 fibrils from their termini and does not attack the fibril at random positions. Nevertheless, the propensity to oligomerization of the WT protein hampered further biophysical studies. A novel stabilized fully human monomeric hRAD51 allowed us to investigate its interaction with BRC4 through orthogonal biophysical methods. SAXS experiments were also carried out on the hRAD51-BRC4 complex to provide novel structural insights on their behavior in complex. Atomistic modelling of generated Alphafold2 models revealed that both proteins display flexible N-terminal domains. These results, along with previous evidence on hRAD51 WT fibrils, suggest that BRC4 binding triggers a conformational rearrangement on the hRAD51 N-terminal domain from a more ordered to an intrinsically disordered state.
2023
4° AIC-BMM
1
35
Rinaldi Francesco, G.S. (2023). Elucidating the BRCA2-RAD51 interaction through an integrated structural biophysics approach.
Rinaldi Francesco, Girotto Stefania, Cavalli Andrea
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/929493
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