Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer
DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double‐strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details o...
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Veröffentlicht in: | Angewandte Chemie International Edition 2023-12, Vol.62 (51), p.e202312517-n/a |
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Zusammenfassung: | DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double‐strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51‐mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.
We report the isolation of a fully human monomeric RAD51, a unique tool for cancer research. Biophysical assays—isothermal titration calorimetry (ITC), fluorescence polarization (FP), and nuclear magnetic resonance (NMR)—allowed evaluating the ability of the protein to retain the binding to BRC4. AlphaFold2 (AF2) modeling of small‐angle X‐ray scattering (SAXS) data revealed novel insights on the RAD51 behavior upon BRC4 binding. |
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ISSN: | 1433-7851 1521-3773 1521-3773 |
DOI: | 10.1002/anie.202312517 |