Application of the methylotrophic yeast sexual mechanism in an
artificial mating type switching system
Hiromi MAEKAWA
Faculty of Agriculture, Kyushu University
Research aims
A number of fungal species, including the two major model yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe, have a homothallic
sexual cycle (self-fertile) while heterothallic species (or strains) need toencounter the same species of the opposite mating type to reproduce [1]. Since
the sexual cycle is generally advantageous for evolution, homothallic strains are
better suited for breeding.
Yeast mating type is determined by the DNA sequence at the MAT genelocus. Mating type switching (MTS) is the mechanism of homothallism in S.
cerevisiae and S. pombe. The molecular mechanism of MTS in S. cerevisiae has

been intensively studied (Fig. 1). In contrast, the methylotrophic yeast Ogataea
polymorpha, which is distantly related to S. cerevisiae, utilizes a different
molecular mechanism to switch mating type [2, 3]. There are two MAT loci, ofwhich only one is transcriptionally active. MTS occurs when the chromosomalpositions of the two MAT loci areswitched by chromosomal inversion
through the inverted repeat (IR)sequences adjacent to the MAT loci [2].

The MTS mechanism in O. polymorphaappears to be simpler than that in S.

cerevisiae and therefore is better suitedasamodelforanartificialMTS

mechanism. This project aimed to identify factors and characteristics of thechromosome that are required for MTS in O. polymorpha.
Methods and Results
In O. polymorpha, MTS requires starvation conditions [2, 3]. This requirement can beovercome by constitutive expression of the transcription factor, Rme1 [4]. Since an inducible
system of MTS is required for more detailed study, an attempt was made to develop aninducible expression system for Rme1 by combining it with the auxin-inducible degron.

Since that did not work well with Rme1, a tetracycline inducible expression system (Tetsystem) was established by transferring the S. cerevisiae system into O. polymorpha, with afew modifications. Next, we attempted to use the Tet system to express RME1.
A candidate approach was taken to identify factors that were necessary for MTS inO. polymorpha, with a particular focus on homologous recombination/repair factors andDNA replication factors. So far, several mutants have been identified as MTS-deficientmutants including rad24, mec3, ddc1, mph1, and mus81, in addition to the previously known
rad51, rad52, and rad17 mutants (Maekawa, unpublished) [5]. ChIP analysis indicated that
Rad52 accumulates at IRs in response to nutritional starvation conditions.
Furthermore, chromosomal elements that play roles in MTS have been investigated.
One of the IR sequences was inverted sothat the repeat sequences make direct
repeats (MAT-DR strain). In theMAT-DR strain, the intervening region
would be looped-out if a recombination
event occurred similar to that at MAT
loci in the wild type (Fig. 2). The
looped-out region was detected in theMAT-DR strain under starvation
conditions, which was dependent on
Mph1 helicase, but not the structure-specific endonuclease Mus81. These results suggest thatthe structure-specific endonuclease was not regulated to induce MTS.
Conclusion
This study identified Ddc1, Mec3, Rad24, Mph1, and Mus81 as MTS factors in addition tothe previously known Rad51, Rad52, and Rad17. Starvation-condition specific accumulationof Rad52 at IRs suggests that a double strand break was induced at or near IRs understarvation conditions. It may be possible that the MTS mechanism induced a crossoverduring repair of the DNA lesion at the IRs. However, MTS deficiency of the mph1 mutant
does not support this hypothesis because Mph1 in S. cerevisiae is known to inhibit
crossovers [6]. Further investigation is required to elucidate the molecular mechanism ofMTS in O. polymorpha.
Analysis of the MAT-DR strain suggests that the orientation of the IRs did not
impact the recombination reaction and the regulation to induce MTS. MAT-DR cells loose
essential gene(s) located within the intervening region when recombination occurs between
repeat sequences, and become inviable. A large-scale screen can be set up to identify more
MTS deficient mutants in the MAT-DR strain, which would enable a better understanding of

the molecular mechanism of MTS. Furthermore, in this study, the tetracycline inducible
expression system was successfully established in O. polymorpha, which will benefit future
research in this species.
References
1) Haber, J. E. (2012) Mating-type genes and MAT switching in Saccharomyces
cerevisiae. Genetics, 191: 33–64.
2) Maekawa, H., Kaneko, Y. (2014) Inversion of the chromosomal region between
two mating type loci switches the mating type in Hansenula polymorpha. PLoS
Genet. 10: e1004796.
3) Hanson, S. J., Byrne, K. P., Wolfe, K. H. (2014) Mating-type switching bychromosomal inversion in methylotrophic yeasts suggests an origin for thethree-locus Saccharomyces cerevisiae system. Proc. Natl. Acad. Sci. U.S.A. 111:E4851–8.
4) Yamamoto, K., Tran, T. N. M., Takegawa, K., Kaneko, Y., Maekawa, H. (2017)
Regulation of mating type switching by the mating type genes and RME1 inOgataea polymorpha. Sci. Rep. 7: 16318.
5) Hanson, S. J., Byrne, K. P., Wolfe, K. H. Flip/flop mating-type switching in the
methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12
pathway. PLoS Genet. 13: e1007092.
6) Prakash R1, Satory D, Dray E, Papusha A, Scheller J, Kramer W, Krejci L, Klein
H, Haber JE, Sung P, Ira G. (2009) Yeast Mph1 helicase dissociates Rad51-madeD-loops: implications for crossover control in mitotic recombination. Genes Dev.
23: 67-79