Biology subjects

Ochsenbein, F.

Publications and source records attributed to Ochsenbein, F..

6 recordsLinked to original sources

bioRxiv2025

Optimized quantitative bacterial two-hybrid (qB2H) for protein-protein interaction assessment

Characterizing mutation effects on protein-protein interactions (PPIs) is crucial for elucidating protein structure and function. Massively parallel PPI variant analyses such as deep mutational scanning (DMS) enable interface identification and generate datasets for machine learning. In cellulo strategies such as two-hybrid systems provide straightforward access to such data, but reliability depends on quantitative properties. Here, we show that existing bacterial two-hybrid (B2H) systems have limitations constraining accurate dataset generation. We engineered and benchmarked optimized quantitative B2H (qB2H) alternatives, enabling strain-independent assays, improved metrics, and generation of high-quality datasets. We demonstrate qB2H utility through interface mapping and binder optimization. Perturbation analysis of single-site variants accurately recovered known ASF1 complex contact positions, matching crystallographic data. Integration of generative AI-based design yielded an ASF1-binding peptide with a 70-fold increase in affinity. qB2H offers to R&D scientists a robust, reusable platform for quantitative PPI analysis, enabling both rational protein engineering and data-driven discovery.

synthetic biology↗

bioRxiv2024

Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting the dissociation of Rad51 filaments by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone, preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and protecting Rad51 filaments from destabilization by Srs2.

molecular biology↗

bioRxiv2023

Disordered regions and folded modules in CAF-1 promote histone deposition in S. pombe

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the S. cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

biochemistry↗

bioRxiv2022

Oct4 interacts with subnucleosomal particles generated by Brg1 at enhancers

BRG1, the catalytic subunit of the mammalian SWI/SNF complexes, is essential for chromatin opening at enhancers. However, the nature of the open chromatin remains unclear. Here we show that in addition to producing histone-free DNA, BRG1 generates hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 base pairs of DNA. Our genome-wide analysis indicates that BRG1 makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude, compared to the region occupied on histone-free DNA. We suggest that BRG1-dependent subnucleosomes orchestrate an epigenetic mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

genomics↗

bioRxiv2021

The Xer activation factor of TLC{Phi} expands the possibilities for Xer recombination

Many mobile elements take advantage of the highly-conserved chromosome dimer resolution system of bacteria, Xer. They participate in the transmission of antibiotic resistance and pathogenicity determinants. In particular, the toxin-linked cryptic satellite phage (TLC{Phi}) plays an essential role in the continuous emergence of new toxigenic clones of the Vibrio cholerae strain at the origin of the ongoing 7th cholera pandemic. The Xer machinery is composed of two chromosomally-encoded tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific 28 base pair site of bacterial chromosomes, dif. The activity of XerD depends on a direct contact with a cell division protein, FtsK, which spatially and temporally constrains the process. TLC{Phi} encodes for a XerD-activation factor (XafT), which drives the integration of the phage into the dif site of the primary chromosome of V. cholerae independently of FtsK. However, XerD does not bind to the attachment site (attP) of TLC{Phi}, which raised questions on the integration process. Here, we compared the integration efficiency of thousands of synthetic mini-TLC{Phi} plasmids harbouring different attP sites and assessed their stability in vivo. In addition, we compared the efficiency with which XafT and the XerD activation domain of FtsK drive recombination reactions in vitro. Taken together, our results suggest that XafT promotes the formation of synaptic complexes between canonical Xer recombination sites and imperfect sites.

microbiology↗

bioRxiv2018

Histone deposition plays an active role in recombination-dependent replication to balance genome2 stability upon replication stress

Replication stress poses a serious threat to genome and epigenome stability. Recombination-Dependent-Replication (RDR) ensures DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways during DNA repair processes, crosstalk coupling RDR and chromatin assembly is largely unexplored. Here, we addressed the contribution of chromatin assembly to replication stress in fission yeast. We expressed a mutated histone (H3-H113D) to genetically impair replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition is required for the completion of RDR. Histone deposition prevents joint-molecules from Rqh1-dependent disassembly, a crosstalk contributing to cell survival upon replication stress but channeling RDR towards deleterious events. Asf1 and CAF-1 act in RDR and CAF-1 recruitment to DNA synthesis associated to RDR requires the HR factor Rad52. Our data establish that CAF-1 counteracts Rqh1 activity at sites of replication stress by promoting repair synthesis-coupled histone deposition. These results demonstrate that histone deposition plays an active role in fine-tuning RDR, a benefit counterbalanced by stabilizing at-risk joint molecules for genome stability.

genetics↗