Biology subjects

Rogers, C. M.

Publications and source records attributed to Rogers, C. M..

8 recordsLinked to original sources

bioRxiv2026

BRCA1 promotes homologous recombination through separable DNA and RAD51 binding activities within its disordered region.

The breast and ovarian tumor suppressor BRCA1 heterodimerizes with BARD1 to promote DNA double-strand break repair by homologous recombination (HR) and to protect stressed DNA replication forks against nuclease attack. The large, intrinsically disordered central region of BRCA1 harbors binding sites for DNA and multiple repair factors, but its lack of stable structure has hindered mechanistic dissection of these activities. Using biochemical mapping and NMR spectroscopy, we delineate the DNA binding and RAD51 interaction interfaces within this region and construct separation-of-function mutants that selectively ablate each activity. Both DNA binding and RAD51 interaction are required for BRCA1-BARD1 to promote RAD51-mediated DNA strand invasion, and DNA binding also contributes to BLM-DNA2 end resection. These findings provide mechanistic insights into how individual ligand binding activities within BRCA1 contribute to genome maintenance.

biochemistry↗

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↗

bioRxiv2021

Genetic modulation of initial sensitivity to Δ9-tetrahydrocannabinol (THC) among the BXD family of mice

Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, {Delta}9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (analgesia/antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.

genetics↗

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↗

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

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↗

bioRxiv2017

Primary souring: a novel bacteria-free method for sour beer production

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term \"primary souring\" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.\n\nChemical compounds studied in this article: Lactic acid (PubChem CID: 612); Ethanol (PubChem CID: 702)\n\nAbbreviations: ABV, alcohol by volume; DIC, differential interference contrast; EtOH, ethanol; FG, final gravity; gDNA, genomic DNA; IBU, international bittering unit; LAB, lactic acid bacteria; LASSO, lactic acid specific soft-agar overlay; N-J, neighbor-joining; OG, original gravity; WLN, Wallerstein Laboratories nutrient; YPD, yeast extract, peptone, and dextrose

microbiology↗