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The helicase/SUMO-targeted ubiquitin ligase Uls1 interacts both physically and functionally with the Holliday junction resolvase Yen1
Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Yen1 is nuclease that can cleave the Holliday junction (HJ), an important DNA intermediate formed during homologous recombination. Here, we show that Yen1 interacts molecularly with Uls1, a SUMO targeted ubiquitin ligase that also belongs to the SWI/SNF-family of DNA-dependent ATPases. We demonstrate that Yen1 is SUMO modified in its carboxyl terminus and that this modification strengthens the interaction between Yen1 and Uls1. Absence of Uls1 increased the steady-state levels of Yen1, but only after extensive DNA damage, suggesting that Uls1 has a role in damage-induced degradation of Yen1. Consistent with a shared role for Uls1 and Yen1, mutations in the two enzymes display similar phenotypes. Both uls1 and yen1 have a negative genetic interaction with the alternative HJ-cleaving nuclease Mus81. This negative genetic interaction is manifested in supersensitivity to DNA damaging agents, but also in a meiotic defect. Neither mus81 uls1 nor mus81 yen1 double mutant diploids can complete meiosis. Moreover, both uls1 and yen1 exacerbates the chromosome mis-segregation phenotype of mus81. However, the mus81 uls1 yen1 triple mutant strain was slightly more sensitive to DNA damage compared to any double mutant combination, indicating that Uls1 and Yen1 also have independent roles in DNA repair. Point mutant alleles of Uls1 (uls1K975R and uls1C1330S/C1333S) that inactivates the ATPase and potential ubiquitin ligase activities are also supersensitive to DNA damage when combined with mus81, indicating that both activities of Uls1 are essential for function. We suggest that Yen1 and Uls1 are involved in an alternative pathway that is responsible for resolving complex DNA repair intermediates in the absence of Mus81.

National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-81361OAI: oai:DiVA.org:su-81361DiVA: diva2:561116
Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-31Bibliographically 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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