Search

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single-celled, model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our three 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 were toxic. We report that this sporadic toxicity affects independently the agar stocks of multiple vendors, has occurred repeatedly over at least three decades, and extends to species in highly diverged taxa. Interestingly, the inhibitory effects displayed variable penetrance and were 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Recombination hotspot-activating DNA sites (e.g., M26 , CCAAT , Oligo-C ) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe . We used a fission yeast-optimized CRISPR-Cas9 system ( SpEDIT ) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6 . PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.

Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the ura4 reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions. Remarkably, every Ura - transformant analyzed had template-directed, PAM-disabling bp substitutions near (5-6 bp away from) the DSB but no DNA site-generating substitutions at distance (42-56 bp). An unsought novel allele, ura4-P127* , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests.

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)., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Protacio, Davidson and Wahls.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

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., (Copyright © 2019 Storey et al.)

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., (Copyright © 2019 Mukiza et al.)

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 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.

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.

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.

Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)(2) tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.

The packaging of DNA in nucleosomes presents obstacles to the action of gene regulatory proteins and polymerases on their natural chromatin substrates. We recently reported that nucleosomes exist in a conformational equilibrium, transiently exposing stretches of their DNA off the histone surface. Such "site exposure" processes potentially provide the needed access of proteins to DNA in chromatin. However, the experiments that reveal site exposure are carried out on timescales of tens of minutes to hours. The actual rates of site exposure are not known. Here we use T7 RNA polymerase and exonuclease III as probes to obtain a more relevant lower bound on the rate of nucleosomal site exposure. We find that the organization of DNA into nucleosomes detectably slows the elongation rate of the polymerase, but that full-length elongation, which requires access to all of the DNA, occurs on the seconds timescale. Independent experiments with exonuclease III, which probes the outermost DNA segments only, similarly show that site exposure in these regions occurs on a timescale of seconds or faster. We conclude that site exposure is sufficiently rapid that it may play a role in the initial binding of regulatory proteins to nucleosomal target sites. These rapid rates argue against a nucleosome sliding model for the mechanism of site exposure. Surprisingly, the measured rates may be too slow to account for the known rates of polymerase elongation in vivo. Mechanisms by which polymerase progression through nucleosomes might be catalyzed are discussed., (Copyright 1997 Academic Press Limited.)

We report the development of an alternative approach to studies on nucleosome transcription in vitro. This model system allows us to follow the real-time, synchronous and single passage of RNA polymerase molecules as they progress through DNA packaged in nucleosomes. Results are obtained using phage T7 RNA polymerase with reconstituted nucleosomes prepared from native histones or from histones in which the two H3 Cys 110 thiol groups have been oxidized to form a disulfide bridge. The lengths and concentrations of radiolabeled transcripts produced as a function of time provide direct measurements of the velocities of transcription on naked DNA and on the nucleosomal particles, and allow both relative and absolute efficiencies of initiation, elongation and completion to be determined. These direct measurements of reveal new features of the elongation process. The velocities of elongation on the nucleosomal templates are slightly but reproducibly slower than those on naked DNA. This difference is found to be due to a slight increase in pausing on the nucleosomal templates. Remarkably, the sites of this increased pausing on the nucleosomal templates are also pause sites on the naked DNA. The velocities of elongation on native or oxidized nucleosomal templates are found to be identical to within +/- 10%. We conclude that nucleosomes having covalently bound H3 molecules are substrates for transcription, suggesting that the splitting of the nucleosome postulated in the lexosome model of nucleosome transcription is not a necessary event. Interesting future applications of this methodology are discussed.