Biology subjects

Wyatt, H. D. M.

Publications and source records attributed to Wyatt, H. D. M..

5 recordsLinked to original sources

bioRxiv2026

Dna2-intrinsic condensation regulates DNA end resection and reveals evolutionary redistribution of condensate grammar

Eukaryotic cells commonly use biomolecular condensation of DNA double-strand break (DSB) repair scaffolds and signaling assemblies to organize repair reactions in space and time. Yet whether DSB end-processing enzymes themselves encode tunable phase separation that potentiates resection remains unclear. Here we show that the long-range resection enzyme Dna2 forms liquid-like condensates through an intrinsically disordered region that is necessary and sufficient for phase separation and catalytic enhancement in Saccharomyces cerevisiae. Dna2 condensates concentrate DNA substrates and enhance end processing, whereas disrupting condensate formation impairs repair kinetics, checkpoint signaling, and chromosome stability. Grafting of the heterologous intrinsically disordered region of human FUS partly rescues condensate formation and function, and Cdk1-dependent phosphorylation sites tune condensate stability and enzymatic output in cis and in trans. Machine-learning-based analysis reveals that condensation-promoting features of fungal Dna2 are shared with a restricted set of human DNA2-associated resection regulators. Together, these findings define phosphorylation-tuned, enzyme-intrinsic phase separation as an organizational principle of DSB end resection while supporting a model in which condensation-promoting features are redistributed among factors operating within conserved genome maintenance pathways.

molecular biology↗

bioRxiv2026

A structure-selective endonuclease drives uniparental mitochondrial DNA inheritance

Maternal inheritance of mitochondrial DNA (mtDNA) is a near-universal feature of eukaryotes1, yet the mechanisms that ensure this by preventing paternal mtDNA inheritance have remained unclear. In both Drosophila and humans, mtDNA is actively eliminated from sperm during spermatogenesis, producing mature sperm whose mitochondria lack their genomes2-5. Here we identify Hotaru, a previously uncharacterized, testis-specific GIY-YIG endonuclease, as a central player in this process. We find that Hotaru is expressed in elongated spermatids, localizes to the mitochondrial matrix, and is required for paternal mtDNA elimination. In hotaru mutants, sperm retain mtDNA at levels comparable to those present before the elimination process. Genetic and biochemical analyses show that Hotaru selectively recognizes and cleaves cruciform DNA structures within the mtDNA control region. Together, these findings identify a dedicated nuclease that enforces mitochondrial genome elimination in the animal male germline and reveal that an unexpected structural feature of mtDNA serves as the molecular determinant of its destruction. By recognizing DNA structure rather than specific sequence motifs, this mechanism is inherently robust to the high mutation rate of mitochondrial genomes.

developmental biology↗

bioRxiv2023

qTAG: An adaptable CRISPR-based endogenous tagging protocol using optimized repair cassettes

Endogenous tagging makes it possible to study a proteins localization, dynamics, and function within its native regulatory context. This is typically accomplished via CRISPR, which involves inserting a sequence encoding a functional tag into the reading frame of a gene. However, this process is often inefficient. Here, we introduce the "quickTAG," or qTAG system, a versatile collection of optimized repair cassettes designed to make CRISPR-mediated tagging more accessible. By including a desired tag sequence linked to a selectable marker in the cassette, integrations can be quickly isolated post-editing. The core sequence scaffold within these constructs incorporates several key features that enhance flexibility and ease of use, such as: specific cassette designs for N- and C-terminus tagging; standardized cloning sequences to simplify the incorporation of homology arms for HDR or MMEJ-based repairs; restriction sites next to each genetic element within the cassette for easy modification of tags and selectable markers; and the inclusion of lox sites flanking the selectable marker to allow for marker gene removal following integration. We showcase the versatility of these cassettes with a diverse range of tags, demonstrating their applications in fluorescence imaging, proximity-dependent biotinylation, epitope tagging, and targeted protein degradation. The adaptability of this scaffold is also exhibited by incorporating novel tags such as mStayGold, which offer enhanced brightness and photostability, reconciling prolonged live-cell imaging of proteins at their endogenous levels. Finally, by leveraging the restriction sites, entirely distinct cassette structures and editing schemes were developed. These enabled scenarios that included conditional expression tagging, selectable knockout tagging, and safe-harbor expression. Our existing and forthcoming collection of plasmids will be accessible through Addgene. It includes ready-to-use constructs targeting common subcellular marker genes, as well as an assortment of tagging cassettes for the tagging of genes of interest. The qTAG system offers an accessible framework to streamline endogenous tagging and will serve as an open resource for researchers to adapt and tailor for their own experiments.

molecular biology↗

bioRxiv2022

Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol

BackgroundOver the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene teraphthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. ResultsIn this study, we used Saccharomyces cerevisiae as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts MHET (monohydroxyethyl terephthalate) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is stable to pH, temperature, and for at least 12 days at room temperature. ConclusionsWe demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will viable alternatives to protein purification-based approaches for plastic degradation.

synthetic biology↗

bioRxiv2022

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide the first comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 237 high-confidence interactors, of which the vast majority represent novel SLX4 binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and novel or emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.

biochemistry↗