Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.12.28.522061

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

Bell, J. C.·Dombrowski, C. C.·Plank, J. L.·Jensen, R. B.·Kowalczykowski, S. C.
View original source ↗Export metadata ↓

Abstract

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individuals lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, Replication Protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins--of which BRCA2 is the defining member in humans --alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to--and the assembly of RAD51 filaments on--a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short pre-assembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation. SignificanceDespite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual RPA-coated ssDNA molecules and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way.

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Bell, J. C., Dombrowski, C. C., Plank, J. L., Jensen, R. B., Kowalczykowski, S. C.. 2022-12-29. BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA. https://doi.org/10.1101/2022.12.28.522061

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗
bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗
bioRxiv2026

Interventional breath analysis reveals oral microbial conversion of 1,2-Propanediol into Propanal and 1-Propanol

Background: Orally exhaled breath contains volatile organic compounds (VOCs) that may reflect local microbial metabolism as well as systemic processes. Consequently, the breath sampling route can influence VOC profiles and complicate biomarker interpretation. Previous studies have shown that 1-propanol is substantially enriched in orally compared with nasally exhaled breath. Objective: To determine whether a controlled oral challenge with 1,2-propanediol (1,2-PD) induces sampling route-dependent changes in breath 1-propanol concentrations. Methods: Ten healthy volunteers received 0.1 mL 1,2-PD diluted in approximately 50 mL water. The solution was used as an oral rinse for approximately 30 s and then expectorated. Matched oral and nasal end-tidal breath samples were collected before administration, immediately afterwards, and at 15-min intervals. Propanal, 1-propanol, acetol, acetone, and isoprene were analysed using gas chromatograph-ion mobility spectrometer (GC-IMS). Results: Oral 1,2-PD exposure produced a rapid and transient increase in exhaled propanal and 1-propanol signals, with the largest responses immediately after administration and at the second post-intervention measurement. In contrast, nasal signals remained comparatively stable. Acetone and isoprene showed no consistent intervention-related changes, regardless of the sampling route. No reproducible increase in acetol was detected, although interpretation was limited by analytical interference. Conclusion: A controlled oral 1,2-PD challenge elicited a route-specific propanal and 1-propanol response, consistent with local microbial metabolism of 1,2-PD within the oral cavity. Both fucose and 1,2-PD have been previously reported in human saliva, including elevated concentrations in selected disease-associated contexts. Bacteria can degrade salivary mucin glycans, releasing sugars including fucose. Together, these observations motivate investigation of a proposed pathway linking mucin-derived fucose to 1,2-PD and subsequent propanal and 1-propanol formation in the human oral cavity.

biochemistry↗