Biology subjects

Girotto, S.

Publications and source records attributed to Girotto, S..

3 recordsLinked to original sources

bioRxiv2025

Breaking new ground into RAD51-BRC repeats interplay in Homologous Recombination

Homologous recombination (HR) is a critical repair pathway involving numerous proteins that ensure error-free DNA double-strand breaks (DSBs) repair. Dysfunction in HR components can compromise genome integrity. Despite advances, many aspects of HR remain poorly understood. Notably, even one of the earliest identified and most critical interactions, between RAD51 and BRCA2, remains incompletely characterized, mainly due to the lack of structural data. This study presents a comprehensive biophysical analysis of the RAD51-BRC repeats interaction, integrating computational and experimental approaches. Starting with assessing the correlation between the binding affinities of individual BRC repeats and their impact on RAD51 disassembly, our investigation extends to larger BRCA2 truncations, offering unprecedented insights into the molecular determinants of RAD51 recognition. As mutations in the BRC repeats impair RAD51 recruitment and are associated with cancer, these results provide a valuable framework for interpreting pathogenic variants and guiding precision medicine therapies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/688182v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@8c5cc9org.highwire.dtl.DTLVardef@11f9066org.highwire.dtl.DTLVardef@14159bdorg.highwire.dtl.DTLVardef@16ac161_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Integrating computational and experimental biophysics reveals novel insights into the RAD51-BRC4 interaction

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/642044v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@32e13org.highwire.dtl.DTLVardef@c60a6forg.highwire.dtl.DTLVardef@1d6fdceorg.highwire.dtl.DTLVardef@1b10541_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG The interaction between the RAD51 and BRCA2 proteins is central for homologous recombination, a crucial pathway ensuring high-fidelity DNA repair. Recruitment of RAD51 involves eight highly conserved regions on BRCA2, named BRC repeats. To date, only the interaction between the fourth BRC repeat (BRC4) and the RAD51 C-terminal domain has been structurally characterized, while the complex of full-length RAD51 with the peptide still remains elusive. Here, we report an integrative experimental and in silico approach to reconstruct the conformational ensemble in solution for full length RAD51 in complex with BRC4. We combined AlphaFold2, crosslinking mass spectrometry (XL-MS) and small angle x-ray scattering (SAXS) data with molecular dynamics simulations (MD). These data show that the full-length RAD51-BRC4 complex is a mixture of compact and elongated conformations. Detailed analysis of the reweighted ensemble, achieved through the maximum entropy principle, identifies key residues at the N-terminal-BRC4 interface mediating complex conformational dynamics. Our evidence provides robust atomic-level insights into the interaction of RAD51 and BRC4. These findings are crucial for understanding the molecular features underlying the recognition between RAD51 and BRCA2, which are essential for developing therapeutic intervention strategies in cancer treatment.

biophysics↗

bioRxiv2021

RAD51 nuclear recruitment and inhibition towards innovative strategies against pancreatic cancer

RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment. Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a domino effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug. The present study sheds further light on the complexity of the HR pathway, paving the way for designing peptide and small organic molecule inhibitors of RAD51 as innovative anticancer and chemo/radiosensitizer compounds.

biophysics↗