Biology subjects

Gray, S. J.

Publications and source records attributed to Gray, S. J..

7 recordsLinked to original sources

bioRxiv2026

Genome-wide mapping of helicase-generated ssDNA reveals Hrq1 activity at RNA polymerase III-transcribed genes

DNA helicases preserve genome stability by unwinding DNA during replication, repair, recombination, and transcription, yet their sites of action in vivo remain difficult to define. Here, we describe a sequencing-based strategy to map helicase activity genome-wide by coupling helicases to the single-stranded DNA-specific activation-induced cytidine deaminase (AID). Deamination of cytosines exposed during helicase-mediated DNA unwinding generates strand-specific mutational footprints that can be detected by whole-genome sequencing at near-nucleotide resolution. Using the Saccharomyces cerevisiae RecQ4-family helicase Hrq1, a functional homolog of human RECQL4, we generated the first genome-wide map of Hrq1 activity. Hrq1-dependent deaminations were highly enriched at RNA polymerase III (RNAPIII)-transcribed genes, particularly tRNA genes, where they occurred predominantly on the transcriptional template strand. This localization was reproducible using both overexpressed Hrq1-AID fusions and an inducible dimerization system that recruited AID to endogenously expressed Hrq1, and it was markedly reduced by helicase-inactivating mutation, indicating that active DNA unwinding underlies the observed signal. Hrq1 associated with nearly all tRNA genes irrespective of transcription level, replication orientation, or proximity to transposable elements, yet deletion or overexpression of Hrq1 did not detectably alter pre-tRNA abundance or RNA polymerase III recycling under the conditions tested. Application of the same approach to the PIF1-family helicase Rrm3 recovered its established enrichment at a subset of highly transcribed, head-on tRNA genes, validating the method. Together, these findings establish AID-mediated mutational footprinting as a general approach for mapping helicase activity in vivo and reveal an unexpected, widespread association of the RecQ4-family helicase Hrq1 with RNAPIII-transcribed genes.

molecular biology↗

bioRxiv2026

FIDDL: depth-matched negative controls distinguish genuine interspecific introgression from competitive-mapping artifact

Interspecific introgression is routinely detected by competitively mapping reads to a concatenated multi-species reference and calling regions where a non-focal species recruits coverage. Using strains that cannot contain the ancestry being detected, we show this design generates substantial false-positive signal through two mechanisms with opposite phylogenetic-distance signatures. Standard nuclear assemblies omit the mitochondrion and 2-micron plasmid, leaving high-copy cytoplasmic reads without a legitimate target; completing the reference preferentially removes signal from the most divergent donor. Genuine cross-species sequence conservation inflates the most closely related donor. Masking chromosome ends removes its subtelomeric part but plateaus at a non-zero floor, and the interior residual traces to conserved single-copy genes where a short read carries under one base of discriminating information. The floor grows with sequencing depth (1.19% of callable positions at 50x, 2.02% at 147x, 3.85% at 393x in a pure strain), is not mitigated by long reads, and appears at sub-diploid dosage - three properties widely read as evidence of authenticity. Because the discriminating information is below single-read resolution, no read-level filter separates artifact from introgression; we show three that fail. What works is locus-level: a consensus-phylogenetic test (29/29 specificity on confirmed artifact) and an allele-fraction donor-match test, complementary and validated in both directions on independent published introgression. We package the comparative controls as FIDDL (False Introgression Detection via Depth-matched controls and Loci-recurrence), an open-source tool, withdraw two of our own analysis-ready calls, and show re-analysis of published wild isolates reduces low-confidence introgression by [~]53% while leaving high-confidence signal intact.

bioinformatics↗

bioRxiv2026

Genome-scale characterization of wild yeasts reveals cryptic diversity and population structure across three genera

Environmental surveys of wild yeasts typically rely on ribosomal barcodes, which cannot resolve cryptic species, interspecific gene flow, mixed cultures, or population structure. To determine what genome-scale characterization adds, we sequenced a representative panel of wild yeasts spanning the genera Saccharomyces, Schizosaccharomyces, and Lachancea using Oxford Nanopore long-read whole-genome sequencing and placed each isolate within published reference datasets. Whole-genome analyses revealed biologically important features that barcoding alone could not detect. A shagbark-hickory isolate resolved as a genuine two-species co-culture. An oak-bark isolate proved to be Schizosaccharomyces versatilis, a recently reinstated species represented by very few known strains, and its analysis demonstrated that standard assembly-quality benchmarks can be misleading for deep-branching taxa. Three Lachancea thermotolerans isolates formed a distinct, previously unsampled population within the wild tree-associated lineage, extending its known geographic range. In contrast, an apparent signal of Saccharomyces eubayanus introgression in two beer-associated S. cerevisiae isolates disappeared after analysis with matched negative controls and de novo assemblies, showing that it reflected mapping artifacts rather than genuine ancestry. Together, these results demonstrate that inexpensive long-read whole-genome sequencing transforms wild-yeast bioprospecting from species identification into a genome-scale framework for resolving cryptic diversity, population structure, and mixed cultures while providing stronger support - and stronger limits - for evolutionary inference. SIGNIFICANCEMost surveys of wild yeasts identify isolates using short DNA barcodes, which are well suited for naming species but often miss the evolutionary relationships and hidden diversity within them. By applying inexpensive whole-genome sequencing to a diverse collection of environmental yeasts, we uncovered previously undetected mixed cultures, a rare recently recognized species, and a distinct wild population, while also showing that an apparent case of interspecies gene exchange was instead a technical artifact. These results demonstrate that genome-scale analysis can both reveal biological diversity that simpler methods overlook and provide the evidence needed to avoid misleading evolutionary conclusions, making it a powerful new approach for studying natural microbial populations.

ecology↗

bioRxiv2026

A genetically buffered helicase network promotes tolerance of G-quadruplex stabilization in Saccharomyces cerevisiae

G-quadruplexes (G4s) are non-canonical DNA secondary structures that can impede DNA replication and transcription and provoke genome instability, and DNA helicases of the PIF1 and RecQ families have long been regarded as the principal enzymes that resolve them. To directly test the relative contributions of these families, we measured the growth of Saccharomyces cerevisiae helicase mutants in the presence of the G4-stabilizing ligand pyridostatin (PDS). Unexpectedly, no single PIF1- or RecQ-family mutant was sensitized to PDS relative to wild type. Sensitivity emerged only in double mutants, and it did so for combinations both within a single family and across the two families. This pattern indicates that G4 tolerance is buffered by the combined, partially interchangeable, activity of multiple helicases rather than by any one family. To ask whether this redundancy extends beyond the canonical players, we tested two additional helicases whose human orthologs are implicated in G4 metabolism: Chl1 (DDX11/ChlR1) and Srs2 (RTEL1). Loss of Chl1 alone did not sensitize cells, and chl1{Delta} combined with PIF1- or RecQ-family mutations recapitulated the redundancy pattern - with one informative exception: chl1{Delta} hrq1{Delta} remained PDS-tolerant, placing Chl1 and Hrq1 in a shared genetic route. In contrast, srs2{Delta} was the sole single mutant sensitized to PDS, defining a non-redundant requirement that no other helicase compensates. We integrate these results into a two-layer model in which a redundant helicase pool resolves G4-associated genomic stress, while a non-redundant Srs2 function manages its recombinogenic consequences. Our findings reframe G4 maintenance from a family-specific activity into a distributed, buffered network. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/737069v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@d3db2borg.highwire.dtl.DTLVardef@d9eebborg.highwire.dtl.DTLVardef@1c868aaorg.highwire.dtl.DTLVardef@4f72d6_HPS_FORMAT_FIGEXP M_FIG C_FIG ARTICLE SUMMARYDNA helicases, enzymes that unwind DNA, are thought to dismantle G-quadruplexes (G4s), four-stranded DNA structures that can block DNA metabolism and destabilize genomes. In Saccharomyces cerevisiae, we used the chemical pyridostatin to stabilize G4s and measured the growth of helicase mutants. Losing any single helicase had no effect, but losing two together - even from different helicase families - impaired growth. The protein Chl1 works with the helicase Hrq1 in one shared pathway, while Srs2 is uniquely required on its own. G4 tolerance therefore depends on a redundant network of helicases. These findings interest researchers studying genome stability and related human cancer-predisposition disorders.

genetics↗

bioRxiv2026

Declaration of Fermentation: Community-Embedded Wild Yeast Bioprospecting as a Model for Place-Based CURE Design

Course-based undergraduate research experiences (CUREs) are widely recognized as a high-impact practice in biology education, yet most existing CURE frameworks treat the research organism as an interchangeable teaching prop rather than a genuine scientific contribution. We argue that place-based, community-embedded CUREs - in which students isolate, characterize, and publicly deploy a locally meaningful wild organism - constitute a qualitatively distinct model warranting broader adoption. As proof of concept, we present the Declaration of Fermentation project at Indiana University Bloomington: graduate researchers isolated a wild Saccharomyces cerevisiae strain from the bark of a campus landmark tree, confirmed its wild provenance by whole-genome sequencing and phylogenomics, and partnered with local craft breweries to produce a colonial-era inspired ale released publicly for the 250th anniversary of the Declaration of Independence. Volunteer sensory panels at two independent public tasting events (combined n = 33-34 per attribute) confirmed a fruity-funky profile consistent with wild-strain fermentation, with no significant differences between events (Mann-Whitney U, Benjamini- Hochberg-corrected p > 0.05 for all 11 attributes). We describe three design principles - genomically confirmed strain identity, mandatory community partnership, and place-based historical narrative - that distinguish this model from prior wild yeast brewing CUREs, discuss how these principles generalize to other institutions and fermentation vehicles, and identify next steps for formal learning assessment. Complete implementation protocols are provided as supplemental Appendices 1-6, and the bioinformatics pipeline is freely available at https://doi.org/10.5281/zenodo.20679384.

microbiology↗

bioRxiv2025

Dimerization of Cdc13 is essential for dynamic DNA exchange on telomeric DNA

Single-stranded DNA (ssDNA) binding proteins (ssBPs) are essential in eukaryotes to protect telomeres from nuclease activity. In Saccharomyces cerevisiae, the ssBP Cdc13 is an essential protein that acts as a central regulator of telomere length homeostasis and chromosome end protection, both alone and as part of the Cdc13-Stn1-Ten1 (CST) complex. Cdc13 has high binding affinity for telomeric ssDNA, with a very slow off-rate. Previously, we reported that despite this tight ssDNA binding, Cdc13 rapidly exchanges between bound and unbound telomeric ssDNA substrates, even at sub-stoichiometric concentrations of competitor ssDNA. This dynamic DNA exchange (DDE) is dependent on the presence and length of telomeric repeat sequence ssDNA and requires both Cdc13 DNA binding domains, OB1 and OB3. Here we investigated if Cdc13 dimerization is important for DDE by characterizing the dimerization mutant Cdc13-L91R. Using mass photometry, we confirmed that Cdc13-L91R fails to dimerize in solution, even in the presence of ssDNA. Gel-based DDE assays revealed that Cdc13-L91R fails to undergo ssDNA exchange compared to recombinant wild-type protein. Biolayer interferometry demonstrated that this effect was not due to differences in ssDNA binding kinetics. Thus, dimerization of Cdc13 is essential for DDE, and we model how this may impact telomere biology in vivo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/645294v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18f34fdorg.highwire.dtl.DTLVardef@e00e71org.highwire.dtl.DTLVardef@1d4400aorg.highwire.dtl.DTLVardef@1b87a7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

In vitro cellular phenotypes of cortical neurons from R255X MECP2 knock-in mice are improved by either expression of wildtype MeCP2 or read-through with G418

Approximately 60% of individuals with Rett syndrome (RTT) carry a nonsense variant in the MECP2 gene; thus, there is an unmet need to identify novel nonsense suppression compound(s) that can restore full length MeCP2 protein levels and function. Here, we characterized neuronal phenotypes in cultured cortical neurons from newborn knock-in mice harboring the MECP2 R255X variant. After 2 weeks in vitro, R255X mutant neurons showed smaller cell bodies, shorter dendrites, fewer dendritic branches, and a lower density of excitatory synapses when compared to wildtype (WT) neurons. Transduction of AAV9-MeCP2-GFP in R255X mutant neurons made these cellular phenotypes similar to those in WT neurons, including soma size, dendritic length and branching, and excitatory synapse density. As proof of principle for the potential clinical use of read-through compounds, cultured R255X mutant neurons treated with the aminoglycoside G418 for 72h in vitro showed cell body size and excitatory synapse density similar to WT neurons. We expect these combined approaches will identify effective compounds to suppress translation termination at a premature termination codon, which can be moved to further preclinical functional and behavioral studies in R255X MECP2 knock-in mice. Summary StatementExpression of wildtype MECP2 or treatment with G418 in vitro restored cell body size, dendritic length, and dendritic spine density in cortical neurons from R255X MECP2 knock-in mice to levels comparable to wildtype neurons.

neuroscience↗