Biology subjects

Bochman, M. L.

Publications and source records attributed to Bochman, M. L..

At least 19 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↗

bioRxiv2026

Vibrio campbellii encodes a distinct set of type III secretion system effectors that mediate cytotoxicity in eukaryotic host models

Type III secretion systems (T3SS) are common virulence factors that facilitate the injection of anti-eukaryotic effector toxins that damage and kill target host cells. The sequence and function of the structural and regulatory proteins of these systems are conserved across Gram-negative pathogens, including Pseudomonas, Yersinia, and Salmonella. However, the identity and function of effector proteins are not conserved and are unknown in several relevant human and animal pathogens. Here, we used comparative genomics to identify and characterize the effectors encoded by Vibrio campbellii BB120, a crustacean and fish pathogen. We showed that most sequenced Vibrio strains belonging to the Harveyi clade, including V. campbellii, encode a full set of structural and regulatory genes corresponding to the T3SS1 of V. parahaemolyticus; the exception is V. natriegens. Transcriptomic and proteomic analyses identified four V. campbellii effectors that are secreted by the T3SS. Among these, two effectors are encoded outside the T3SS island, and all effector genes were co-regulated by both the master T3SS regulator ExsA and by the master quorum sensing regulator LuxR. Three effectors - VopS, CopA, and VIBHAR_06684 - exhibited toxic activity in yeast cells or bone marrow-derived macrophages, and the toxicity phenotypes were dependent on a functional T3SS. VopS and CopA are conserved among the queried species of the Harveyi clade. VIBHAR_06684 or VIBHAR_05674 did not show conservation among the queried species. These findings demonstrate that T3SSs in bacteria from the same clade have conserved structural secretion apparatuses but exhibit variance in effector repertoires. We postulate that the functions of effectors differ between species to impart roles in host specificity. Author SummaryPathogenic Vibrio bacteria employ many virulence factors to cause disease in marine animals, including biofilms, proteases, and the secretion of toxins. Here, we identify four secreted toxins encoded by V. campbellii that are toxic to eukaryotic cells. These toxins are secreted by the type III secretion system, a needle-like apparatus used by many bacteria to inject toxins into host cells. The toxins identified in this study are regulated by quorum sensing, a method bacteria use to communicate and control virulence genes required for pathogenesis. The results of this study indicate that different Vibrio species employ distinct sets of toxins that possibly facilitate their interaction with a host.

microbiology↗

bioRxiv2026

DNA binding converts an inactive RecQ4-family helicase into a dominant-negative DNA repair factor

DNA inter-strand crosslinks (ICLs) are highly cytotoxic lesions that require coordinated processing for repair. The RecQ4-family helicase Hrq1 promotes ICL repair in Saccharomyces cerevisiae, yet the mechanistic basis of its function remains unclear. Notably, the catalytically inactive hrq1-K318A allele confers greater sensitivity to ICL-inducing agents than deletion of HRQ1, suggesting a dominant-negative effect. To define the basis of this phenotype, we performed a genetic suppressor screen combined with biochemical and structural analyses. Spontaneous suppressors of hrq1-K318A sensitivity were overwhelmingly intragenic second-site mutations, many of which are predicted to destabilize the protein or impair its ability to bind DNA. In all cases, these mutations alleviated the dominant-negative repair defect. Biochemical characterization of representative mutants, including a rationally designed DNA-binding mutant, demonstrated that disruption of DNA binding suppresses hrq1-K318A toxicity even when protein stability is retained. These findings support a model in which DNA engagement by a catalytically inactive RecQ4-family helicase contributes to dominant-negative interference with DNA repair. More broadly, this work provides insight into how incomplete loss-of-function alleles of human RECQL4 may disrupt genome maintenance pathways. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/705743v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@82adb8org.highwire.dtl.DTLVardef@15b1fcforg.highwire.dtl.DTLVardef@1855d10org.highwire.dtl.DTLVardef@a104ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A screen for synthetic genetic interactions with the Saccharomyces cerevisiae hrq1ΔN allele

The Saccharomyces cerevisiae Hrq1 helicase is a functional homolog of the disease-linked human RECQL4 enzyme and has been used as a model to study RecQ4 helicase subfamily biology. Although the motor cores of Hrq1 and RECQL4 are quite similar, these proteins display distinct N-terminal domains (NTDs) of unknown function. Do these domains facilitate species-specific activities by the two helicases, or do they serve common roles despite their differences in sequence and predicted structure? We probed these questions here by analyzing an NTD-truncated isoform of Hrq1 (Hrq1{Delta}N) both in vitro and in vivo. We found that the Hrq1 NTD houses a cryptic DNA binding site and that the hrq1{Delta}N allele is distinct from both hrq1{Delta} and the catalytically inactive hrq1-K318A mutant. Using synthetic genetic array analysis of hrq1{Delta}N crossed to the yeast S. cerevisiae single-gene deletion and temperature-sensitive allele collections, we also identified hundreds of synthetic genetic interactions. As with similar analyses of hrq1{Delta} and hrq1-K318A, our results suggest roles for Hrq1 and its NTD in multiple physiological pathways that underpin genome integrity. Together, these data are guiding our ongoing efforts to understand the roles of Hrq1 and RECQL4 in genome maintenance, which will help to explain why RECQL4 mutations cause disease. ARTICLE SUMMARYThis work should interest researchers in the genome integrity and yeast disease model fields. It continues ongoing efforts to develop the Saccharomyces cerevisiae Hrq1 helicase as a model to understand the disease-linked human RECQL4 helicase. Hrq1 and RECQL4 share similar helicase cores but have divergent N-terminal domains (NTDs) of unknown function. The results demonstrate that the Hrq1 NTD contains a DNA binding site, and an HRQ1 allele lacking its NTD (hrq1{Delta}N) interacts with hundreds of mutants in defined allele collections. Thus, the hrq1{Delta}N allele is distinct from the previously characterized hrq1{Delta} and hrq1-K318A (catalytically inactive) mutants.

molecular biology↗

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↗

bioRxiv2023

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA

Telomerase is the enzyme that lengthens telomeres and is tightly regulated by a variety of means to maintain genome integrity. Several DNA helicases function at telomeres, and we previously found that the Saccharomyces cerevisiae helicases Hrq1 and Pif1 directly regulate telomerase. To extend these findings, we are investigating the interplay between helicases, single-stranded DNA (ssDNA) binding proteins (ssBPs), and telomerase. The yeast ssBPs Cdc13 and RPA differentially affect Hrq1 and Pif1 helicase activity, and experiments to measure helicase disruption of Cdc13/ssDNA complexes instead revealed that Cdc13 can exchange between substrates. Although other ssBPs display dynamic binding, this was unexpected with Cdc13 due to the reported in vitro stability of the Cdc13/telomeric ssDNA complex. We found that the DNA exchange by Cdc13 occurs rapidly at physiological temperatures, requires telomeric repeat sequence DNA, and is affected by ssDNA length. Cdc13 truncations revealed that the low-affinity binding site (OB1), which is distal from the high-affinity binding site (OB3), is required for this intermolecular dynamic DNA exchange (DDE). We hypothesize that DDE by Cdc13 is the basis for how Cdc13 "moves" at telomeres to alternate between modes where it regulates telomerase activity and assists in telomere replication.

biochemistry↗

bioRxiv2021

Mixed culture metagenomics of the microbes making sour beer

Mixed microbial cultures create sour beers, but many brewers do not know which microbes comprise their cultures. The objective of this work was to use deep sequencing to identify microorganisms in sour beers brewed by spontaneous and non-spontaneous methods. Twenty samples were received from brewers, which were processed for microbiome analysis by next generation sequencing. For bacteria, primers were used to amplify the V3-V4 region of the 16S rRNA gene; fungal DNA detection was performed using primers to amplify the entire internal transcribed spacer region. The sequencing results were then used for taxonomy assignment, sample composition, and diversity analyses, as well as nucleotide BLAST searching. We identified 60 genera and 140 species of bacteria, of which the most prevalent were Lactobacillus acetotolerans, Pediococcus damnosus, and Ralstonia picketti/mannitolilytica. In fungal identification, 19 genera and 26 species were found, among which the most common yeasts were Brettanomyces bruxellensis and Saccharomyces cerevisiae. In some cases, genetic material from more than 60 microorganisms was found in a single sample. In conclusion, we were able to determine the microbiomes of various mixed cultures used to produce beer, providing useful information to better understand the sour beer fermentation process and brewing techniques.

microbiology↗

bioRxiv2021

Isolation of wild yeasts from Olympic National Park and Moniliella megachiliensis ONP131 physiological characterization for beer fermentation

Thousands of yeasts have the potential for industrial application, though many were initially considered contaminants in the beer industry. However, these organisms are currently considered important components in beers because they contribute new flavors. Non-Saccharomyces wild yeasts can be important tools in the development of new products, and the objective of this work was to obtain and characterize novel yeast isolates for their ability to produce beer. Wild yeasts were isolated from environmental samples from Olympic National Park and analyzed for their ability to ferment malt extract medium and beer wort. Six different strains were isolated, of which Moniliella megachiliensis ONP131 displayed the highest levels of attenuation during fermentations. We found that M. megachiliensis could be propagated in common yeast media, tolerated incubation temperatures of 37{degrees}C and a pH of 2.5, and was able to grow in media containing maltose as the sole carbon source. Yeast cultivation was considerably impacted (p<0.05) by lactic acid, ethanol, and high concentrations of maltose, but ONP131 was tolerant to high salinity and hop acid concentrations. This is one of the first physiological characterizations of M. megachiliensis, which has potential for the production of beer and other fermented beverages.

microbiology↗

bioRxiv2020

Comprehensive synthetic genetic array analysis of alleles that interact with mutation of the Saccharomyces cerevisiae RecQ helicases Hrq1 and Sgs1

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1{Delta} and sgs1{Delta} mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1{Delta}, sgs1{Delta}, hrq1-K318A, sgs1-K706A, hrq1{Delta} sgs1{Delta}, hrq1{Delta} sgs1-K706A, hrq1-K318A sgs1{Delta}, and hrq1-K318A sgsl-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

genetics↗

bioRxiv2020

The genetic and physical interactomes of the Saccharomyces cerevisiae Hrq1 helicase

The human genome encodes five RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) that participate in various processes underpinning genomic stability. Of these enzymes, the disease-associated RECQL4 is comparatively understudied due to a variety of technical challenges. However, Saccharomyces cerevisiae encodes a functional homolog of RECQL4 called Hrq1, which is more amenable to experimentation and has recently been shown to be involved in DNA inter-strand crosslink (ICL) repair and telomere maintenance. To expand our understanding of Hrq1 and the RecQ4 subfamily of helicases in general, we took a multi-omics approach to define the Hrq1 interactome in yeast. Using synthetic genetic array analysis, we found that mutations of genes involved in processes such as DNA repair, chromosome segregation, and transcription synthetically interact with deletion of HRQ1 and the catalytically inactive hrq1-K318A allele. Pull-down of tagged Hrq1 and mass spectrometry identification of interacting partners similarly underscored links to these processes and others. Focusing on transcription, we found that hrq1 mutant cells are sensitive to caffeine and that mutation of HRQ1 alters the expression levels of hundreds of genes. In the case of hrq1-K318A, several of the most highly upregulated genes encode proteins of unknown function whose expression levels are also increased by DNA ICL damage. Together, our results suggest a heretofore unrecognized role for Hrq1 in transcription, as well as novel members of the Hrq1 ICL repair pathway. These data expand our understanding of RecQ4 subfamily helicase biology and help to explain why mutations in human RECQL4 cause diseases of genomic instability.

genetics↗

bioRxiv2020

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

DNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae, a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2- mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulates Pso2 translesion nuclease activity through a site- specific ICL in vitro. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions.

biochemistry↗

bioRxiv2018

An organoleptic survey of meads made with lactic acid-producing yeasts

We previously reported the isolation a suite of wild lactic acid-producing yeasts (LAYs) that enable "primary souring" during beer fermentation without the use of lactic acid bacteria. With sour meads gaining popularity in modern mead making, we were interested in exploring the same primary souring approach to traditional semi-sweet meads. In this study, we utilized 13 LAY strains to produce semi-sweet meads using a standardized batch of honey must to ensure consistent starting conditions. Thirteen 11-L batches of mead were prepared, and each was inoculated with one of the LAY strains, along with two control batches inoculated with champagne yeast. The initial pH and specific gravity were measured for each batch before inoculation. Traditional organic staggered nutrient addition was utilized for the first 72 h of fermentation with specific gravities being taken throughout the mead making process. Meads were racked, tasted, stabilized, cold crashed, bottled, and transported to the American Mead Makers Association 2018 Conference in Broomfield, Colorado. There, organoleptic surveys were conducted on these meads utilizing an array of tasters with varying levels of mead sensory analysis experience. The results of the sensory analysis, focusing on aroma and flavor, are discussed.

microbiology↗

bioRxiv2018

The S. cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro

Telomere length homeostasis is vital to maintaining genomic stability and is regulated by multiple factors, including telomerase activity and DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RecQ-like helicase 4 (RECQL4), plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an in vitro primer extension assay, here we determined the effects of recombinant wild-type and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity depending on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis in vivo. We propose a model in which Hrq1 and Pif1 cooperatively contribute to telomere length homeostasis in yeast.

biochemistry↗