The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination

Abstract

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a “domino” mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer

“…The detailed mechanism of RAD51 fibril disassembly was recently described as a multistep process, in which BRC4 erodes RAD51 fibrils from their termini through a “domino” mechanism [13] . This yields monomeric RAD51, which is the first essential step in BRCA2‐mediated homologous recombination [13] . AF2 predicted a conformational shift of RAD51 N‐terminal when it is in complex with BRC4 (Figure 7).…”

“…It was recently demonstrated that BRC4 can disassemble RAD51 fibrils in the absence of DNA. [13] This study suggested that, upon DNA damage, BRCA2 (through its BRC repeats) disassembles RAD51 cytosolic fibrils, recruits monomers of RAD51, and transports them into the nucleus. [13] The oligomeric behavior of RAD51 makes it challenging to investigate this mechanism, allowing for the discovery of only scraps of indirect biochemical evidence of RAD51's interaction with the BRC repeats.…”

“…[13] This study suggested that, upon DNA damage, BRCA2 (through its BRC repeats) disassembles RAD51 cytosolic fibrils, recruits monomers of RAD51, and transports them into the nucleus. [13] The oligomeric behavior of RAD51 makes it challenging to investigate this mechanism, allowing for the discovery of only scraps of indirect biochemical evidence of RAD51's interaction with the BRC repeats. [14,15] Disruption of the BRCA2-RAD51 interaction has emerged as a promising strategy for synthetic lethality, including in the combination with PARP inhibitors (PARPi).…”

“…Indeed, this N-terminal unlocking mechanism is in line with BRC4 mechanism of fibrils erosion from their termini that yields to monomeric RAD51. [13] To verify this hypothesis, the His-RAD51-His-BRC4 complex was co-expressed and co-purified to perform SEC-SAXS experiments (Figures S26, S27, and Table S2). Primary data analysis confirmed protein molecular weight and provided a R g of 34 Å (Table S2).…”

Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition

“…RAD51 is a highly structurally conserved molecule in organisms ranging from Escherichia coli to higher mammals and plays an important role in maintaining genome stability [1,2]. RAD51 forms a complex with breast cancer susceptibility gene 2 (BRCA2) upon DNA damage, such as DNA double-strand breaks (DSB) for homologous recombination (HR) repair [3,4]. HR is catalyzed by RAD51 recombinase, which forms a nucleoprotein filament on resected single-stranded DNA (ssDNA) at the damage site, and mediates the pairing of homologous DNA sequences and strand invasion [5].…”