Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii).

The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies and functional analyses of centromere DNA elements, and has been shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation-inducing conditions, showing that the switching system was totally non-functional. The hoΔstrains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔstrains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for performing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii.

Several sources of DNA damage compromise the integrity and stability of the genome of every organism. Specifically, DNA double-strand breaks (DSBs) can have lethal consequences for the cell. To repair this type of DNA damage, the cells employ homology-directed repair pathways or non-homologous end joining. Homology-directed repair requires the activity of the RAD52 epistasis group of genes. Rad52 is the main recombination protein in the budding yeast Saccharomyces cerevisiae, and rad52Δ mutants have been characterized to show severe defects in DSB repair and other recombination events. Here, we identified the RAD52 gene in the budding yeast Naumovozyma castellii. Our analysis showed that the primary amino acid sequence of N. castellii Rad52 shared 70% similarity with S. cerevisiae Rad52. To characterize the gene function, we developed rad52Δ mutant strains by targeted gene replacement transformation. We found that N. castellii rad52Δ mutants showed lowered growth capacity, a moderately altered cell morphology and increased sensitivity to genotoxic agents. The decreased viability of the N. castellii rad52Δ mutants in the presence of genotoxic agents indicates that the role of the Rad52 protein in the repair of DNA damage is conserved in this species.

Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii.

The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of ∼275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.

New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation.

Telomerase maintains telomere length and chromosome integrity by adding short tandem repeats of single-stranded DNA to the 3' ends, via reverse transcription of a defined template region of its RNA subunit. To further understand the telomerase elongation mechanism, we studied the primer utilization and extension activity of the telomerase from the budding yeast Naumovozyma castellii (Saccharomyces castellii), which displays a processive nucleotide and repeat addition polymerization. For the efficient initiation of canonical elongation, telomerase required 4-nt primer 3' end complementarity to the template RNA. This DNA-RNA hybrid formation was highly important for the stabilization of an initiation-competent telomerase-DNA complex. Anchor site interactions with the DNA provided additional stabilization to the complex. Our studies indicate three additional separate interactions along the length of the DNA primer, each providing different and distinct contributions to the initiation event. A sequence-independent anchor site interaction acts immediately adjacent to the base-pairing 3' end, indicating a protein anchor site positioned very close to the catalytic site. Two additional anchor regions further 5' on the DNA provide sequence-specific contributions to the initiation of elongation. Remarkably, a non-telomeric sequence in the distal 25- to 32-nt region negatively influences the initiation of telomerase elongation, suggesting an anchor site with a regulatory role in the telomerase elongation decision.

Metabolomic charactetization of yeast cells after dehydration stress.

In this study, we analyzed the metabolite features of the yeasts Saccharomyces cerevisiae, Naumovia castellii, and Saccharomyces mikatae. The three species are closely related genetically but differ in their tolerance of desiccation stress. Specifically, we determined whether certain metabolites correlated with cell viability after stress imposition. The metabolomics profiles of these strains were compared before cell desiccation and after cell rehydration. In S. mikatae, the presence of lysine or glutamine during rehydration led to a 20% increase in survival whereas during dehydration the levels of both amino acids in this yeast were drastically reduced.