Biology subjects

Ferlez, B.

Publications and source records attributed to Ferlez, B..

5 recordsLinked to original sources

bioRxiv2025

Evolution of Eukaryotic Specific DNA Binding Sites in Asgard Archaeal RecA Recombinases

Homologous recombination (HR) relies on the RecA-family recombinases Rad51 and Dmc1, which both catalyze DNA strand pairing and exchange but promote distinct recombination outcomes. How these recombinases became functionally specialized remains unclear. Here, we combine phylogenetic analysis with functional assays to define the basis of this specialization. We identify conserved amino acid variation in the DNA binding Loop 2 region across the archaeal eukaryotic lineage and show that these residues tune recombinase interactions with double-stranded DNA. Guided by these evolutionary signatures, we engineer Rad51 variants with altered DNA binding properties and test their functions. Rad51 like variants promote break-induced replication (BIR), whereas substitutions characteristic of Dmc1 suppress BIR and instead favor crossover outcomes, as measured using mitotic crossover assays. These results indicate that evolutionary divergence in Loop 2 alters recombinase binding on reaction intermediates, influencing their stability and progression toward distinct repair outcomes. Thus, evolutionary tuning of recombinase DNA interactions shift the balance between recombination pathways, providing a mechanistic basis for the functional specialization of Rad51 and Dmc1 during mitotic and meiotic HR.

evolutionary biology↗

bioRxiv2024

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

microbiology↗

bioRxiv2024

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA template for repair during the homology search stage of HR. Failure to execute the homology search quickly and efficiently can result in complex intermediates that generate genomic rearrangements, a hallmark of human cancers. Rad54 is an ATP dependent DNA motor protein that functions during the homology search by regulating the recombinase Rad51. How this regulation reduces genomic rearrangements is currently unknown. To better understand how Rad54 can prevent genomic rearrangements, we evaluated several amino acid mutations in Rad54 that were found in the COSMIC database. COSMIC is a collection of amino acid mutations identified in human cancers. These substitutions led to reduced Rad54 function and the discovery of a conserved motif in Rad54. Through genetic, biochemical, and single-molecule approaches, we show that disruption of this motif leads to failure in stabilizing early strand invasion intermediates, causing loss-of-heterozygosity rearrangements. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. This mechanism is likely fundamental to eukaryotic biology.

biochemistry↗

bioRxiv2023

Rad53 phosphorylation of Rdh54 regulates the lifetime of homologous recombination intermediates

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 regulates Rdh54 clustering activity as revealed by single molecule imaging. This stems from phosphorylation dependent and independent interactions between Rdh54 and Rad53. Genetic assays reveal that loss of phosphorylation leads to phenotypic changes resulting in loss-of-heterozygosity (LOH) outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.

biochemistry↗

bioRxiv2022

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair the damaged recipient DNA, HR requires an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulating this transfer of information is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH) in diploid organisms, and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR but has a limited role in repairing DNA. Instead, several existing phenotypes in rdh54{Delta} strains suggest that Rdh54 may regulate the flow of information between donor and recipient DNA molecules post DNA repair. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information after DNA repair. Our data indicates that RDH54 regulates DNA sequence exchange between chromosomes by limiting the disruption of Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 also protects Rad51 filaments, acting in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, displays a different distribution of information exchange outcomes than rdh54{Delta} cells. From these results, we propose a model for how Rdh54 may effectively regulate information transfer during homologous recombination.

biochemistry↗