| 1. |
- Cohn, Marita, et al.
(författare)
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Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii
- 2019
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Ingår i: G3: Genes, Genomes, Genetics. - : Oxford University Press (OUP). - 2160-1836. ; 9:10, s. 3345-3358
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Tidskriftsartikel (refereegranskat)abstract
- 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 toensuretelomere function,collectively known asAlternative Lengthening ofTelomeres(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 telomericsequence(ITS)functionsastheinitiationpointfortheadditionoftheTelKOelement.This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.
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| 2. |
- Karademir Andersson, Ahu, et al.
(författare)
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Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii)
- 2016
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 33:12, s. 633-646
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Tidskriftsartikel (refereegranskat)abstract
- The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was 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 hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains 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 doing 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.
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| 3. |
- Karademir Andersson, Ahu, et al.
(författare)
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Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation
- 2017
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 429:14, s. 2109-2123
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Karademir Andersson, Ahu, et al.
(författare)
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Naumovozyma castellii: an alternative model for budding yeast molecular biology
- 2017
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 34:3, s. 95-109
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Tidskriftsartikel (refereegranskat)abstract
- Naumovozyma castellii (Saccharomyces castellii) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained an increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Due to the suitable phylogenetic distance to S. cerevisiae the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii, focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution.
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| 5. |
- Karademir Andersson, Ahu
(författare)
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Studies of molecular mechanisms of telomere maintenance in Naumovozyma castellii
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Telomeres are special DNA-protein structures that protect ends of chromosomes from being recognized as double-strand breaks. Telomeres consist of tandemly repeated units of TG-rich DNA and associate with telomere-specific proteins.When a cell replicates its chromosomes, a certain piece of DNA is lost from chromosomal ends due to the ´´ end replication problem´´. This progressive shortening of telomeric sequences leads to inability of the chromosomal ends to distinguish themselves from double-stranded breaks. When a cell reaches to this crisis point, its proliferation will stop, leading to replicative senescence and eventually apoptosis. The most common way to counteract telomere loss is utilization of telomerase enzyme. Telomerase extends telomeres by using its intrinsic RNA template. Telomerase is active in human germ cells, cancer cells and certain eukaryotic species including the budding yeast Naumovozyma castellii. Most cancers cells (85-90 %) gain cellular immortality by re-activating the telomerase enzyme. The remaining cancer cells use alternative ways to restore lengths of their telomeres.In my doctoral studies I investigated molecular mechanisms of telomere maintenance in N. castellii. We reviewed the historical background of its establishment as a model organism and summarized its significant contributions to understanding of various molecular biological pathways. We developed stable haploid strains, which are amenable for genetic studies, to study telomerase-independent telomere maintenance. We characterized N. castellii telomerase regarding to its substrate specificity and priming capacity. We proposed a model where four different interactions occur, influencing the initiation of its priming capacity. We have showed the anchoring DNA regions that influence the initiation of telomere extension. Lastly, we investigated how telomeres are maintained when telomerase is disabled. Surprisingly, telomerase-negative cells proliferate without a detectable growth crisis. We showed that they maintain a short stretch of telomeric seqeunce at the very ends of chromosomes and also wild type structural organization at the chromosomal ends.In conclusion, I investigated the telomere maintenance from two different perspectives. These parallel studies contributed to understanding of the dynamics of telomere maintenance. Moreover, they emphasized conserved features of telomere biology, helping visualizing the evolutionary origins of telomerase and maintenance of linear chromosomes.
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