DNA double-strand breaks (DSBs) are primarily repaired in eukaryotic cells through two pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The thermotolerant yeast Kluyveromyces marxianus is recognized for its highly active NHEJ pathway, making it a suitable model organism for studying the role of NHEJ in DSB repair. To induce DSBs in K. marxianus DMKU3-1042, an expression cassette containing the gene encoding the endonuclease RsaI was integrated into the LYS1 locus of both the wild-type and NHEJ-deficient KU70 mutant strains. This cassette is regulated by the galactose-inducible promoter GAL10. Cells expressing RsaI and grown in galactose medium exhibited an elongated, rod-shaped morphology under a microscope. Following RsaI expression, the viability of transformed KU70 cells decreased during the first three hours of culture in liquid medium and then partially recovered after six hours of incubation. In contrast, the KU70 mutant cells failed to produce viable survivors. Pulsed-field gel electrophoresis analysis revealed distinct chromosomal separation patterns among various RsaI-transformed KU70 cells. These findings demonstrate that the repair of RsaI-induced DSBs in K. marxianus DMKU3-1042 results in new strains with several forms of rearranged chromosomes.
Abdel-Banat et al. (2026) studied this question.