A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis.
Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.
Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Jiang
J
Chen
Stefan U
SU
Aström
D020134:Catalytic Domain; D003434:Crossing Over, Genetic; D004249:DNA Damage; D059766:DNA End-Joining Repair; D045566:DNA, Cruciform / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042822:Genomic Instability; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D008540:Meiosis / Q000235:genetics; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D045566:DNA, Cruciform; D005656:Fungal Proteins; D045565:Holliday Junction Resolvases; D053484:RecQ Helicases