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Regulation and mechanism of mating-type switching in Kluyveromyces lactis
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transposable elements (TEs) have had immense impact on the structure, function and evolution of eukaryotic genomes. The work in this thesis identified Kat1, a novel domesticated DNA transposase of the hAT family in the yeast Kluyveromyces lactis. Kat1 triggers a genome rearrangement that results in a switch of mating type from MATa to MATα. Furthermore, Kat1 acts on sequences that presumably are ancient remnants of a long-lost transposable element. Therefore, Kat1 provides a remarkable example of the intricate relationship between transposable elements and their hosts. We showed that Kat1 generates two DNA double strand breaks (DSBs) in MATa and that the DDE motif and several other conserved amino acid residues are important for Kat1 cleavage activity. DNA hairpins were formed on one end of the DSBs whereas the DNA between the DSBs was joined into a circle. Kat1 was transcriptionally activated by nutrient limitation through the transcription factor Mts1 and negatively regulated by translational frameshifting. In conclusion, Kat1 is a highly regulated domesticated transposase that induces sexual differentiation. 

In another study, we developed an assay to measure switching rates in K. lactis and found that the switching rate was ~6x10-4 events/generation. In a genetic screen for mutations that increased mating-type switching, we found mutations in the RAS1 gene. The small GTPase Ras1 regulates cellular cyclic AMP levels and we demonstrated that Mts1 transcription is regulated by the RAS/cAMP pathway and the transcription factor Msn2. Since Ras activity is regulated by nutrient availability, these data likely explains why nutrient limitation induces mating-type switching.

 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2015. , 62 p.
Keyword [en]
yeast, mating-type switching, DSB, gene conversion, transposable elements, RAS/cAMP pathway, nutrient limitation
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-117671ISBN: 978-91-7649-204-8 (print)OAI: oai:DiVA.org:su-117671DiVA: diva2:815256
Public defence
2015-09-01, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2015-06-11 Created: 2015-05-29 Last updated: 2015-06-11Bibliographically approved
List of papers
1. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast
Open this publication in new window or tab >>Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast
2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 43, 15491-15496 p.Article in journal (Refereed) Published
Abstract [en]

Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATa in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1-MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HML alpha) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MAT alpha locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed -1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATa genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase- derived endonuclease vital for sexual differentiation.

Keyword
mating type, DNA double-strand break, transposable element, frameshift, DNA hairpin
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-109969 (URN)10.1073/pnas.1406027111 (DOI)000343729500060 ()
Note

AuthorCount:4;

Available from: 2014-12-04 Created: 2014-12-02 Last updated: 2017-12-05Bibliographically approved
2. RAS/Cyclic AMP and Transcription Factor Msn2 Regulate Mating and Mating-Type Switching in the Yeast Kluyveromyces lactis
Open this publication in new window or tab >>RAS/Cyclic AMP and Transcription Factor Msn2 Regulate Mating and Mating-Type Switching in the Yeast Kluyveromyces lactis
2011 (English)In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 10, no 11, 1545-1552 p.Article in journal (Refereed) Published
Abstract [en]

In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis.

National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-65652 (URN)10.1128/EC.05158-11 (DOI)000296723600019 ()21890818 (PubMedID)
Available from: 2011-12-13 Created: 2011-12-13 Last updated: 2017-12-08Bibliographically approved

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