Mechanism for inverted-repeat recombination induced by a replication fork barrier

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

A systematic genome-wide account of binding sites for the model transcription factor Gcn4

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method ("G-SELEX"), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

Engineering of Saccharomyces cerevisiae for 24-Methylene-Cholesterol Production

24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in Saccharomyces cerevisiae by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast’s native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms—including the DHCR7 from Physalis angulata (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from Oryza sativa and XlDHCR7 from Xenopus laevis—were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the XlDHCR7 expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.

Chromosome Genome Sequencing and Comparative Transcriptome-Based Analyses of Kloeckera apiculata 34-9 Unveil the Potential Biocontrol Mechanisms Against Citrus Green Mold

Biological control is an environmentally friendly, safe, and replaceable strategy for disease management. Genome sequences of a certain biocontrol agent could lay a solid foundation for the research of molecular biology, and the more refined the reference genome, the more information it provides. In the present study, a higher resolution genome of Kloeckera apiculata 34-9 was assembled using high-throughput chromosome conformation capture (Hi-C) technology. A total of 8.07 M sequences of K. apiculata 34-9 genome was anchored onto 7 pesudochromosomes, which accounting for about 99.51% of the whole assembled sequences, and 4,014 protein-coding genes were annotated. Meanwhile, the detailed gene expression changes of K. apiculata 34-9 were obtained under low temperature and co-incubation with Penicillium digitatum treatments, respectively. Totally 254 differentially expressed genes (DEGs) were detected with low temperature treatment, of which 184 and 70 genes were upregulated and downregulated, respectively. Some candidate genes were significantly enriched in ribosome biosynthesis in eukaryotes and ABC transporters. The expression of gene Kap003732 and Kap001595 remained upregulated and downregulated through the entire time-points, respectively, indicating that they might be core genes for positive and negative response to low temperature stress. When co-incubation with P. digitatum, a total of 2,364 DEGs were found, and there were 1,247 upregulated and 1,117 downregulated genes, respectively. Biosynthesis of lysine and arginine, and phenylalanine metabolism were the highest enrichment of the cluster and KEGG analyses of the co-DEGs, the results showed that they might be involved in the positive regulation of K. apiculata 34-9 response to P. digitatum. The completeness of K. apiculata 34-9 genome and the transcriptome data presented here are essential for providing a high-quality genomic resource and it might serve as valuable molecular properties for further studies on yeast genome, expression pattern of biocontrol system, and postharvest citrus storage and preservation.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

Genome-wide distribution of Rad26 and Rad1-Rad10 reveals their relationship with Mediator and RNA polymerase II

Transcription is coupled with DNA repair, especially within nucleotide excision repair (NER). Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and NER via a direct contact with Rad2/XPG endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/CSB and that of Rad1-Rad10/XPF-ERCC1, addressing the question on a potential interplay of these proteins with Mediator and Pol II in yeast Saccharomyces cerevisiae. Our genome-wide analyses show that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially on highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II. Moreover, we revealed that Rad1-Rad10 and Rad26 colocalize with Mediator on intergenic regions and physically interact with this complex. Using kin28 TFIIH mutant, we showed that Mediator stabilization on core promoters lead to an increase in Rad1-Rad10 chromatin binding, whereas Rad26 occupancy is less impacted by Mediator and follows mainly a decrease in Pol II transcription. Combined with multivariate analyses, our results reveal the interplay between Rad1-Rad10, Rad26, Mediator and Pol II, modulated by the binding dynamics of Mediator and Pol II transcription. In conclusion, we extend the Mediator link to Rad1-Rad10 and Rad26 NER proteins and reveal important differences in Mediator relationships with Rad2, Rad1-Rad10 and Rad26. Our work thus contributes to new concepts of the functional interplay between transcription and DNA repair, relevant for human diseases including cancer and XP/CS syndromes.

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.