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

Telomere length homeostasis is vital for 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 WT 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

Two Novel Strains of Torulaspora delbrueckii Isolated from the Honey Bee Microbiome and Their Use in Honey Fermentation

Yeasts are ubiquitous microbes found in virtually all environments. Many yeast species can ferment sugar into ethanol and CO2, and humans have taken advantage of these characteristics to produce fermented beverages for thousands of years. As a naturally abundant source of fermentable sugar, honey has had a central role in such fermentations since Neolithic times. However, as beverage fermentation has become industrialized, the processes have been streamlined, including the narrow and almost exclusive usage of yeasts in the genus Saccharomyces for fermentation. We set out to identify wild honey- or honey-bee-related yeasts that can be used in honey fermentation. Here, we isolated two strains of Torulaspora delbrueckii from the gut of a locally collected honey bee. Both strains were able to ferment honey sugar into mead but failed to metabolize more than a modest amount of wort sugar in trial beer fermentations. Further, the meads fermented by the T. delbrueckii strains displayed better sensory characteristics than mead fermented by a champagne yeast. The combination of T. delbrueckii and champagne yeast strains was also able to rapidly ferment honey at an industrial scale. Thus, wild yeasts represent a largely untapped reservoir for the introduction of desirable sensory characteristics in fermented beverages such as mead

The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologues. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work

Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA

Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel DNA patrolling activity which may underlie Pif1’s diverse functions: a Pif1 monomer preferentially anchors itself to a 3′-tailed DNA junction and periodically reel in the 3′ tail with a step size of one nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex but not duplex DNA. Instead of leaving after G4 unwinding, allowing it to refold, or going beyond to unwind duplex DNA, Pif1 repeatedly unwinds G4 DNA, keeping it unfolded. Pif1-induced unfolding of G4 occurs in three discrete steps, one strand at a time, and is powerful enough to overcome G4-stabilizing drugs. The periodic patrolling activity may keep Pif1 at its site of in vivo action in displacing telomerase, resolving R-loops, and keeping G4 unfolded during replication, recombination and repair

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications

ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing1. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization2,3, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms4. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au–thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(i)–thiolates that localize in the nucleus5. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA