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.

Genetic and biochemical interactions of yeast DNA helicases.

DNA helicases function in many types of nucleic acid transactions, and as such, they are vital for genome integrity. Although they are often considered individually, work from many groups demonstrates that these enzymes often genetically and biochemically interact in vivo. Here, we highlight methods to interrogate such interactions among the PIF1 (Pif1 and Rrm3) and RecQ (Hrq1 and Sgs1) family helicases in Saccharomyces cerevisiae. The interactions among these enzymes were investigated in vivo using deletion and inactivation alleles with a gross-chromosomal rearrangement (GCR) assay. Further, wild-type and inactive recombinant proteins were used to determine the effects of the helicases on telomerase activity in vitro. We found that synergistic increases in GCR rates often occur in double vs. single mutants, suggesting that the helicases function in distinct genome integrity pathways. Further, the recombinant helicases can function together in vitro to modulate telomerase activity. Overall, the data suggest that the interactions among the members of these DNA helicase families are multipartite and argue for a comprehensive systems biology approach to fully elucidate the physiological interplay between these enzymes.

Pif1 Activity is Modulated by DNA Sequence and Structure.

The gene encoding the Pif1 helicase was first discovered in a Saccharomyces cerevisiae genetic screen as a mutant that reduces recombination between mitochondrial respiratory mutants and was subsequently rediscovered in a screen for genes affecting the telomere length in the nucleus. It is now known that Pif1 is involved in numerous aspects of DNA metabolism. All known functions of Pif1 rely on binding to DNA substrates followed by ATP hydrolysis, coupling the energy released to translocation along DNA to unwind duplex DNA or alternative DNA secondary structures. The interaction of Pif1 with higher-order DNA structures, like G-quadruplex DNA, as well as the length of single-stranded (ss)DNA necessary for Pif1 loading have been widely studied. Here, to test the effects of ssDNA length, sequence, and structure on Pif1's biochemical activities in vitro, we used a suite of oligonucleotide-based substrates to perform a basic characterization of Pif1 ssDNA binding, ATPase activity, and helicase activity. Using recombinant, untagged S. cerevisiae Pif1, we found that Pif1 preferentially binds to structured G-rich ssDNA, but the preferred binding substrates failed to maximally stimulate ATPase activity. In helicase assays, significant DNA unwinding activity was detected at Pif1 concentrations as low as 250 pM. Helicase assays also demonstrated that Pif1 most efficiently unwinds DNA fork substrates with unstructured ssDNA tails. As the chemical step size of Pif1 has been determined to be 1 ATP per translocation or unwinding event, this implies that the highly structured DNA inhibits conformational changes in Pif1 that couple ATP hydrolysis to DNA translocation and unwinding.

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.

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 Maker's 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.

Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice.

Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

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.

Convergent donor and acceptor substrate utilization among kinase ribozymes.

Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2'OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2'OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson-Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that-unlike most aptamers-these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.

Bulk phase biochemistry of PIF1 and RecQ4 family helicases.

DNA helicases are involved in nearly all facets of genome integrity, and in humans, mutations in helicase-encoding genes are often linked to diseases of genomic instability. Two highly studied and evolutionarily conserved helicase families are the PIF1 and RecQ helicases. Enzymes in these families have known roles in DNA replication, recombination, and repair, as well as telomere maintenance, DNA recombination, and transcription. Although genetics, structural biology, and a variety of other techniques have been used to study these helicases, ensemble analyses of their basic biochemical activities such as DNA binding, ATP hydrolysis, and DNA unwinding have made significant contributions to our understanding of their physiological roles. Here, we present general methods to generate recombinant proteins from both helicase families, as well as standard biochemical assays to investigate their activities on DNA.

Characterization of the telomerase modulating activities of yeast DNA helicases.

Eukaryotes with linear chromosomes circumvent the end replication problem via the action of a specialized ribonucleoprotein reverse transcriptase known as telomerase. Cells lacking telomerase activity will senesce when their chromosome ends shorten to a critical length. In contrast, cancer cells can become immortalized by upregulating telomerase to lengthen telomeres during each cycle of DNA replication. Thus, the regulation of telomerase is critical for normal telomere homeostasis. Of the various known ways that telomerase activity is modulated in vivo, recent studies have demonstrated that DNA helicases are involved. In Saccharomyces cerevisiae, the Hrq1 and Pif1 helicases act in a pathway that regulates telomerase extension at telomeres and at DNA double-strand DNA breaks. In vitro analysis demonstrates that when these helicases are combined in reactions, they synergistically inhibit or stimulate telomerase activity depending on which helicase is catalytically active. Here, we describe the methods for the overproduction and purification of Hrq1 and Pif1. We also report the preparation of partially purified cell extracts with telomerase activity and how the effects of these helicase on telomerase activity can be assessed in vitro.

The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain.

: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.

HIV-1 reverse transcriptase pausing at bulky 2' adducts is relieved by deletion of the RNase H domain.

Pausing by reverse transcriptase (RT) during retroviral replication increases the frequency of homologous strand transfer, nucleotide misincorporation, and non-templated nucleotide addition. Pausing frequency increases at sites of DNA damage or upon incorporation of nucleotide analogs with steric barriers. These lesions thus likely stimulate mutations leading to resistant viral strains that escape drug treatments or immune surveillance. To study the response of retroviral RTs to bulky 2' adducts, a ribozyme-catalyzed reaction was used to generate an RNA template strand containing a thiophosphate adduct at a specific 2'-hydroxyl located upstream from a polyadenosine sequence. Subsequent alkylation increased the size of the adduct. Polymerization readthrough efficiencies were compared for mature RTs derived from HIV-1 (p66/p51), AMV (p95/p63), MMLV (p80 monomer), and a truncated version of HIV-1 RT lacking the RNase H domain (p51/p51 homodimer). Readthrough at the 2' lesion was markedly greater for the p51/p51 homodimer of HIV-1 RT than for the other enzymes, suggesting that the presence of the RNase H domain increases the probability that the modified primer/template will encounter a barrier to translocation. Comparison to published structures suggests potential unfavorable interactions between the 2' adduct and W24, F61, I63, D76, and R78 in the fingers domain of the RT. We propose that the enhanced readthrough observed upon RNase H domain deletion alters the trajectory of the primer/template in this region that diminishes steric and electrostatic clash with these residues. The template also included a penta-adenosine sequence that induced pausing in the order MMLV > HIV-1 (p66/p51) > AMV ~ HIV-1 (p51/p51).

Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

Structural and functional analyses of stem-loop 1 of the Sindbis virus genome.

Alphavirus genome function is controlled by elements at both the 5' and 3' ends. The 5' 220 nt of the Sindbis virus genome is predicted to consist of four stem-loop structures the first of which has been demonstrated to be required for efficient minus-strand RNA synthesis. To understand the role of the structure of the first stem-loop (SL1) in regulating genome function, we performed enzymatic and chemical probing analyses. There were significant differences between the computer-predicted structures and our experimental data. In the 5' terminus, two loop regions appear to be interacting in a complex and interdependent fashion with non-Watson-Crick interactions involving multiple adenosine residues playing a critical role in determining the overall structure. Some of the mutations that disrupted these interactions had significant affects, both positive and negative, on minus-strand synthesis, and translational efficiency was generally increased. In the context of full-length virus, these structural changes resulted in reduced virus growth kinetics particularly in mosquito cells suggesting host-specific effects of mutations in this region of the viral genome. Possible SL1 structures based on our experimental data are discussed.

A trans acting ribozyme that phosphorylates exogenous RNA.

The structural complexity required for substrate recognition within an active site constrains the evolution of novel catalytic functions. To evaluate those constraints within populations of incipient ribozymes, we performed a selection for kinase ribozymes under conditions that allowed competition for phosphorylation at nine candidate sites. Two candidate sites are the hydroxyl groups on a "quasi-diffusible" chloramphenicol (Cam) moiety tethered to the evolving library through an inert, flexible linker. A subtractive step was included to allow only seven ribose 2' hydroxyls to compete with the two Cam hydroxyls for phosphorylation. After the library was incubated with gamma-thio-ATP (ATPgammaS), active species were recovered from a polyacrylamide gel containing [(N-acryloylamino)phenyl] mercury (APM) and amplified for further cycles of selection. Activity assays on selected isolates and truncated derivatives identified the essential secondary structure of the dominant RNA motif. Phosphorylation was independent of the Cam moiety, indicating ribose 2' phosphorylation. The dominant motif was separated into catalytic "ribozyme" and "substrate" strands. Partial alkaline digestion of the substrate strand before and after phosphorylation identified the precise modification site as the first purine (R) within the required sequence 5'-RAAAANCG-3'. The reaction shows approximately 10-fold preference for ATPgammaS over ATP and is independent of pH over a wide range (5.5-8.9), consistent with a dissociative reaction mechanism that is rate-limited by formation of a metaphosphate transition state. Divalent metal ions are required, with a slight preference of Mn(2+) > Mg(2+) > Ca(2+). Lack of reactivity in [Co(NH(3))(6)](3+) indicates a requirement for inner sphere contact with the metal ion, either for structural stabilization, catalysis, or both.

Expressing RNA aptamers inside cells to reveal proteome and ribonome function.

Small RNA molecules can participate in complex regulatory networks not merely through Watson-Crick interactions with other RNAs (as in anti-sense RNA), but also by forming discrete structures that bind protein or low-molecular weight targets. RNA aptamers derived from in vitro selections (SELEX) are being used inside cells for at least four purposes: (1) to antagonise normal cellular proteins as a means of elucidating their biological roles; (2) as decoys to natural RNA-binding proteins to reveal their functions or those of structural elements within naturally occurring transcripts; (3) as regulatory modules to govern the expression of exogenous genes; and (4) to antagonise disease-related targets for potential biomedical applications. This paper summarises recent advances in each of these areas, the parameters that influence successful application and the potential for future developments. The use of aptamers in vivo may serve as a paradigm for understanding how some non-coding RNAs and other elements of the ribonome exert their influence on the intracellular proteome, and is thus becoming an important tool for modern cell and molecular biology.

Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli.

A better understanding of aptamer function in bacteria would help to establish simple model systems for screening RNA-protein interactions within an intracellular context. Escherichia coli DNA polymerase I mutants (Pol I(ts)) fail to grow at 37 degrees C unless an exogenous DNA polymerase such as HIV-1 reverse transcriptase (RT) is expressed within the cell. Here, we show that four RNA aptamers that inhibit HIV-1 RT in vitro block complementation by HIV-1 RT when expressed in vivo. No other essential functions are impaired by aptamer expression at either temperature. Intracellular aptamer RNA concentrations from induced cultures were measured to range from 76 to 180 nM, which is comparable with exogenously expressed HIV-1 RT levels in these cells. RT polymerase activity was reduced to background levels in cell-free extracts prepared from cultures expressing both HIV-1 RT and the 70.28 aptamer, compared with extracts from cultures expressing HIV-1 RT alone. Intracellularly expressed RNA aptamers can thus be used to generate conditional null mutants in bacteria by titrating an essential protein.