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A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis
Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
2012 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 11, no 10, 833-843 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2012. Vol. 11, no 10, 833-843 p.
Keyword [en]
Yen1, Holliday junction, DNA repair, Yeast, Homologous recombination, Replication fork
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-81358DOI: 10.1016/j.dnarep.2012.07.004ISI: 000310761100006OAI: oai:DiVA.org:su-81358DiVA: diva2:561109
Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2017-12-07Bibliographically approved
In thesis
1. DNA double-strand break repair in ascomycetes
Open this publication in new window or tab >>DNA double-strand break repair in ascomycetes
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are two pathways for DNA double strand break (DSB) repair. We found that the NHEJ protein Nej1 interacted physically with the HR protein Srs2, which was dependent on phosphorylation of Nej1 by Dun1. Srs2 recruitment to a DSB partly relied on Nej1 and Dun1. Both Nej1 and Srs2 contributed to efficient single strand annealing (SSA). We suggest that Nej1 and Srs2 facilitate SSA-like repair by disassembling Rad51 nucleoprotein filaments.

Yen1 is a nuclease that can cleave branched recombination intermediates such as Holliday junctions (HJs). We demonstrated that yen1Δ displayed a negative genetic interaction with mus81 and sgs1 mutants. Mus81 and Sgs1 promoted HJ disjoining by alternative routes, explaining the genetic interaction. Interestingly, catalytically inactive Yen1 had residual functions in DNA repair, suggesting that Yen1 also has a structural role. We discovered that Yen1 interacted physically with Uls1 a potential SUMO targeted ubiquitin ligase. The interaction partly depended on SUMO-modification of the carboxyl terminus of Yen1 and consistent with an ubiquitin ligase function for Uls1, absence of Uls1 stabilized Yen1 after extensive DNA damage. In addition, uls1Δ shared several phenotypes with yen1Δ, including negative genetic interactions with Mus81 after DNA damage and in meiosis. We suggest that Yen1 and Uls1 act together in a DNA repair pathway that is responsible for resolving complex repair intermediates in the absence of Mus81.

We found that phosphorylation of histone H2A serine 129 promoted DSB repair. Moreover, cells lacking acetylation of lysine residues in the histone H3 NH2-terminus was defective for HR. Interestingly; leaving a single lysine residue intact protected cells from DNA damage. These findings indicate that both histone H2A phosphorylation and histone H3 acetylation are important for the efficiency of the HR-pathway probably by increasing the accessibility of chromatin.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2012. 53 p.
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-81089 (URN)978-91-7447-580-7 (ISBN)
Public defence
2012-11-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2012-10-25 Created: 2012-10-09 Last updated: 2013-04-08Bibliographically approved

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