Expression of Endonuclease RsaI Induces Chromosomal Rearrangement in the Yeast Kluyveromyces marxianus

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 <i>Kluyveromyces marxianus</i> 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 <i>K. marxianus</i> DMKU3-1042, an expression cassette containing the gene encoding the endonuclease <i>Rsa</i>I was integrated into the <i>LYS1</i> locus of both the wild-type and NHEJ-deficient <i>KU70</i> mutant strains. This cassette is regulated by the galactose-inducible promoter <i>GAL10</i>. Cells expressing <i>Rsa</i>I and grown in galactose medium exhibited an elongated, rod-shaped morphology under a microscope. Following <i>Rsa</i>I expression, the viability of transformed <i>KU70</i> cells decreased during the first three hours of culture in liquid medium and then partially recovered after six hours of incubation. In contrast, the <i>KU70</i> mutant cells failed to produce viable survivors. Pulsed-field gel electrophoresis analysis revealed distinct chromosomal separation patterns among various <i>Rsa</i>I-transformed <i>KU70</i> cells. These findings demonstrate that the repair of <i>Rsa</i>I-induced DSBs in <i>K. marxianus</i> DMKU3-1042 results in new strains with several forms of rearranged chromosomes.