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Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 29, 12037-12042 p.Article in journal (Refereed) Published
Abstract [en]

Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

Place, publisher, year, edition, pages
2009. Vol. 106, no 29, 12037-12042 p.
Keyword [en]
nonhomologous end joining, single strand annealing
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-25628DOI: 10.1073/pnas.0903869106.ISI: 000268178400041OAI: oai:DiVA.org:su-25628DiVA: diva2:200090
Available from: 2008-11-25 Created: 2008-11-17 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Characterization of Budding Yeast Nonhomologous End-Joining at DNA Double-Strand Breaks and Telomeres
Open this publication in new window or tab >>Characterization of Budding Yeast Nonhomologous End-Joining at DNA Double-Strand Breaks and Telomeres
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The yeast K. lactis efficiently integrates DNA by illegitimate recombination (IR). IR was completely dependent upon nonhomologous end-joining (NHEJ). In contrast to S. cerevisiae, NHEJ in K. lactis repaired a wide variety of DNA ends, and was not regulated by cell-type. IR events occurred primarily in 5’ intergenic regions. Deletion of RAD52 did not affect the distribution of IR sites. IR events could be targeted to an ectopic DNA double-strand break (DSB). We interpret the genomic distribution of IR events as a map of mitotic DSBs.

K. lactis mre11 and rad50 strains displayed a shortened telomere phenotype, and a ku80 strain displayed shortened telomeres within a background of telomere elongation. Deletion of KU80 also resulted in excess 3’ overhangs, indicative of a role in telomere end protection. Substantial increases in subtelomeric recombination were observed in rad50 and mre11 strains, while ku80 and lig4 strains displayed modest increases. Telomere-telomere fusions (T-Tfs) induced by loss of Rap1 binding at telomeres were dependent on LIG4 and KU80, but occurred independently of NEJ1. Circularized chromosomes severely inhibited passage of a diploid strain through meiosis. We conclude that K. lactis NHEJ proteins both mediate T-Tfs and contribute to telomere capping.

The Srs2 helicase was identified as a Nej1 interaction partner. Mutational analysis of Nej1 suggested that the interaction was dependent on phosphorylation of Nej1 serines 297/298 by the Dun1 kinase. Deletion of NEJ1 or DUN1 impaired Srs2 recruitment to an HO induced DSB. Srs2 recruitment was unaffected by deletion of SIZ1, implicated in promoting Srs2 recruitment to replication forks. Both srs2 and nej1 strains were deficient in a RAD52-dependent repair event similar to single-strand annealing. Additionally, srs2 and dun1 strains performed NHEJ less efficiently than a wild-type strain. We propose that Nej1 promotes Srs2 recruitment to DSBs, and supports efficient NHEJ/SSA by antagonizing Rad51.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2008. 134 p.
Keyword
DNA repair, nonhomologous end-joining, double-strand break
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-8329 (URN)978-91-7155-787-2 (ISBN)
Public defence
2008-12-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-11-25 Created: 2008-11-17Bibliographically approved
2. 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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