Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge.

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5' single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5' most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

Laboratory horror stories: Poison in the agars.

The fission yeast Schizosaccharomyces pombe is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Two residues in the DNA binding site of Pif1 helicase are essential for nuclear functions but dispensable for mitochondrial respiratory growth.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.

DNA sequences and distinct mechanisms for ura4-595 and ura4-294 alleles of S. pombe.

The ura4 gene of the fission yeast Schizosaccharomyces pombe supports both positive and negative selection; consequently, this gene is widely employed as a powerful tool to study diverse biological processes. Here we report the DNA sequences of two functionally null alleles, ura4-595 and ura4-294 . The ura4-595 allele has a four bp duplication of bp +63 to +66 (5'-CAAG-3') within the ORF and the ura4-294 allele has a nonsynonymous substitution (G to A) at bp +679. We infer that these alleles arose, respectively, by DNA polymerase template slipping and by nucleotide misincorporation (likely via cytosine deamination).

Adaptive Control of the Meiotic Recombination Landscape by DNA Site-dependent Hotspots With Implications for Evolution.

Meiosis is an essential component of the sexual life cycle in eukaryotes. The independent assortment of chromosomes in meiosis increases genetic diversity at the level of whole chromosomes and meiotic recombination increases genetic diversity within chromosomes. The resulting variability fuels evolution. Interestingly, global mapping of recombination in diverse taxa revealed dramatic changes in its frequency distribution between closely related species, subspecies, and even isolated populations of the same species. New insight into mechanisms for these evolutionarily rapid changes has come from analyses of environmentally induced plasticity of recombination in fission yeast. Many different DNA sites, and where identified their binding/activator proteins, control the positioning of recombination at hotspots. Each different class of hotspots functions as an independently controlled rheostat that modulates rates of recombination over a broad dynamic range in response to changing conditions. Together, this independent modulation can rapidly and dramatically alter the global frequency distribution of recombination. This process likely contributes substantially to (i.e., can largely explain) evolutionarily rapid, Prdm9-independent changes in the recombination landscape. Moreover, the precise control mechanisms allow cells to dynamically favor or disfavor newly arising combinations of linked alleles in response to changing extracellular and intracellular conditions, which has striking implications for the impacts of meiotic recombination on evolution.

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination.

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags.

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity. We implemented and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine time points. We report that basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS3. We quantified 2275 proteins across three biological replicates and found that differential expression peaked near 90 min following heat shock (with 868 differentially expressed proteins at 5% false discovery rate). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

Targeted Forward Genetics: Population-Scale Analyses of Allele Replacements Spanning Thousands of Base Pairs in Fission Yeast.

Precise allele replacement (genome editing), without unwanted changes to the genome, provides a powerful tool to define the functions of DNA elements and encoded factors in their normal biological context. While CRISPR is now used extensively for gene targeting, its utility for precise allele replacement at population scale is limited because: (A) there is a strict requirement for a correctly positioned PAM motif to introduce recombinogenic dsDNA breaks (DSBs); (B) efficient replacements only occur very close to the DSBs; and (C) indels and off-target changes are frequently generated. Here we show, using a saturated mutation library with about 15,000 alleles of the ade6 gene of Schizosaccharomyces pombe, that pop-in, pop-out allele replacement circumvents these problems. Two rounds of selection ensure that clones arise by homologous recombination with the target locus. Moreover, the exceptionally high efficiency allows one to carry out the process in bulk, then screen individual clones for phenotypes and genotypes. Alleles were introduced successfully throughout the region targeted, up to 1,956 base pairs from the DSB. About 11% of mutant alleles were hypomorphic, demonstrating utility for analyses of essential genes and genetic elements. This process of "targeted forward genetics" can be used to analyze comprehensively, across thousands of base pairs within a specific target region, a variety of allelic changes, such as scanning amino acid substitutions, deletions, and epitope tags. The overall approach and optimized workflow are extensible to other organisms that support gene targeting.

Diverse DNA Sequence Motifs Activate Meiotic Recombination Hotspots Through a Common Chromatin Remodeling Pathway.

In meiosis, multiple different DNA sequence motifs help to position homologous recombination at hotspots in the genome. How do the seemingly disparate cis-acting regulatory modules each promote locally the activity of the basal recombination machinery? We defined molecular mechanisms of action for five different hotspot-activating DNA motifs (M26, CCAAT, Oligo-C, 4095, 4156) located independently at the same site within the ade6 locus of the fission yeast Schizosaccharomyces pombe Each motif promoted meiotic recombination (i.e., is active) within this context, and this activity required the respective binding proteins (transcription factors Atf1, Pcr1, Php2, Php3, Php5, Rst2). High-resolution analyses of chromatin structure by nucleosome scanning assays revealed that each motif triggers the displacement of nucleosomes surrounding the hotspot motif in meiosis. This chromatin remodeling required the respective sequence-specific binding proteins, was constitutive for two motifs, and was enhanced meiotically for three others. Hotspot activity of each motif strongly required the ATP-dependent chromatin remodeling enzyme Snf22 (Snf2/Swi2), with lesser dependence on Gcn5, Mst2, and Hrp3. These findings support a model in which most meiotic recombination hotspots are positioned by the binding of transcription factors to their respective DNA sites. The functional redundancy of multiple, sequence-specific protein-DNA complexes converges upon shared chromatin remodeling pathways that help provide the basal recombination machinery (Spo11/Rec12 complex) access to its DNA substrates within chromatin.

Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot.

BACKGROUND: Meiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome. Recombination occurs within and is regulated in part by chromatin structure, but relatively few of the many chromatin remodeling factors and histone posttranslational modifications (PTMs) have been interrogated for a role in the process. RESULTS: We developed a chromatin affinity purification and mass spectrometry-based approach to identify proteins and histone PTMs that regulate recombination hotspots. Small (4.2 kbp) minichromosomes (MiniCs) bearing the fission yeast ade6-M26 hotspot or a basal recombination control were purified approximately 100,000-fold under native conditions from meiosis; then, associated proteins and histone PTMs were identified by mass spectrometry. Proteins and PTMs enriched at the hotspot included known regulators (Atf1, Pcr1, Mst2, Snf22, H3K14ac), validating the approach. The abundance of individual histones varied dynamically during meiotic progression in hotspot versus basal control MiniCs, as did a subset of 34 different histone PTMs, implicating these as potential regulators. Measurements of basal and hotspot recombination in null mutants confirmed that additional, hotspot-enriched proteins are bona fide regulators of hotspot activation within the genome. These chromatin-mediated regulators include histone H2A-H2B and H3-H4 chaperones (Nap1, Hip1/Hir1), subunits of the Ino80 complex (Arp5, Arp8), a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex (Swr1, Swc2), and a nucleosome evictor (Fft3/Fun30). CONCLUSIONS: Overall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and histone PTMs participate in designating where meiotic recombination occurs in the genome, and they provide new insight into molecular mechanisms of the process.

In Vivo Metabolic Tracing Demonstrates the Site-Specific Contribution of Hepatic Ethanol Metabolism to Histone Acetylation.

BACKGROUND: Epigenetic dysregulation through ethanol (EtOH)-induced changes in DNA methylation and histone modifications has been implicated in several alcohol-related disorders such as alcoholic liver disease. EtOH metabolism in the liver results in the formation of acetate, a metabolite that can be converted to acetyl-CoA, which can then be used by histone acetyltransferases to acetylate lysine residues. EtOH metabolism in the liver can also indirectly influence lysine acetylation through NAD+ -dependent sirtuin activity that is altered due to increases in NADH. As a proof-of-concept study to determine the direct influence of hepatic EtOH metabolism on histone acetylation changes, we used heavy-labeled EtOH (13 C2 ) and mass spectrometry (MS) to site specifically characterize lysine acetylation on histone proteins. METHODS: Eight-week-old male C57BL/6J mice were gavaged using a bolus dose of either 13 C2 -labeled EtOH (5 g/kg) or maltose dextrin. Blood and livers were collected at 0, 4, and 24 hours followed by histone protein enrichment and derivatization using acid extraction and propionylation, respectively. Metabolic tracing and relative quantitation of acetylated histone proteins were performed using a hybrid quadrupole-orbitrap mass spectrometer. Data were analyzed using MaxQuant, Xcalibur Qual Browser, and the Bioconductor package "mzR." The contribution of EtOH to histone acetylation was quantified using the change in relative abundance of stable isotope incorporation in acetylated peptides detected by MS. RESULTS: Data show significant incorporation of the EtOH-derived 13 C2 -label into N-terminal lysine acetylation sites on histones H3 and H4 after 4 hours, with rapid turnover of labeled histone acetylation sites and return to endogenous levels at 24 hours postgavage. Moreover, site-specific selectivity was observed in regard to label incorporation into certain lysine acetylation sites as determined by tandem mass spectrometry and comparison to isotope simulations. CONCLUSIONS: These data provide the first quantitative evidence of how hepatic EtOH metabolism directly influences histone lysine acetylation in a site-specific manner and may influence EtOH-induced gene expression through these transcriptionally activating chromatin marks.

The NIH must reduce disparities in funding to maximize its return on investments from taxpayers.

New data from the NIH reveal that the scientific return on its sponsored research reaches a maximum at around $400,000 of annual support per principal investigator. We discuss the implications of this 'sweet spot' for funding policy, and propose that the NIH should limit both the minimum and maximum amount of funding per researcher.

Biases in grant proposal success rates, funding rates and award sizes affect the geographical distribution of funding for biomedical research.

The ability of the United States to most efficiently make breakthroughs on the biology, diagnosis and treatment of human diseases requires that physicians and scientists in each state have equal access to federal research grants and grant dollars. However, despite legislative and administrative efforts to ensure equal access, the majority of funding for biomedical research is concentrated in a minority of states. To gain insight into the causes of such disparity, funding metrics were examined for all NIH research project grants (RPGs) from 2004 to 2013. State-by-state differences in per application success rates, per investigator funding rates, and average award size each contributed significantly to vast disparities (greater than 100-fold range) in per capita RPG funding to individual states. To the extent tested, there was no significant association overall between scientific productivity and per capita funding, suggesting that the unbalanced allocation of funding is unrelated to the quality of scientists in each state. These findings reveal key sources of bias in, and new insight into the accuracy of, the funding process. They also support evidence-based recommendations for how the NIH could better utilize the scientific talent and capacity that is present throughout the United States.

Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3).

In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI (+)] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.

A CRISPR-based approach for proteomic analysis of a single genomic locus.

Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome. However, defining local epiproteomes has been limited by a lack of effective technologies to isolate discrete sections of chromatin and to identify with precision specific proteins and histone posttranslational modifications (PTMs). We report the use of the Cas9 and guide RNA (gRNA) components of the CRISPR system for gRNA-directed purification of a discrete section of chromatin. Quantitative mass spectrometry provides for unambiguous identification of proteins and histone PTMs specifically associated with the enriched chromatin. This CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach revealed changes in the local epiproteome of a promoter during activation of transcription. CRISPR-ChAP-MS thus has broad applications for discovering molecular components and dynamic regulation of any in vivo activity at a given chromosomal location.

Binding of the transcription factor Atf1 to promoters serves as a barrier to phase nucleosome arrays and avoid cryptic transcription.

Schizosaccharomyces pombe displays a large transcriptional response common to several stress conditions, regulated primarily by the transcription factor Atf1. Atf1-dependent promoters contain especially broad nucleosome depleted regions (NDRs) prior to stress imposition. We show here that basal binding of Atf1 to these promoters competes with histones to create wider NDRs at stress genes. Moreover, deletion of atf1 results in nucleosome disorganization specifically at stress coding regions and derepresses antisense transcription. Our data indicate that the transcription factor binding to promoters acts as an effective barrier to fix the +1 nucleosome and phase downstream nucleosome arrays to prevent cryptic transcription.

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe.

Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.