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Characterization of Budding Yeast Nonhomologous End-Joining at DNA Double-Strand Breaks and Telomeres
Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
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 [en]
DNA repair, nonhomologous end-joining, double-strand break
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-8329ISBN: 978-91-7155-787-2 (print)OAI: oai:DiVA.org:su-8329DiVA: diva2:200091
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
List of papers
1. Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis
Open this publication in new window or tab >>Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis
2006 In: Nucleic Acids Research, ISSN 1362-4962, Vol. 34, no 5, 1633-1645 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25626 (URN)
Note
Part of urn:nbn:se:su:diva-8329Available from: 2008-11-25 Created: 2008-11-17Bibliographically approved
2. The Role of Nonhomologous End-Joining Components in Telomere Metabolism in Kluyveromyces lactis
Open this publication in new window or tab >>The Role of Nonhomologous End-Joining Components in Telomere Metabolism in Kluyveromyces lactis
Show others...
2007 In: Genetics, ISSN 1091-6490, Vol. 175, no 3, 1035-1045 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25627 (URN)
Note
Part of urn:nbn:se:su:diva-8329Available from: 2008-11-25 Created: 2008-11-17Bibliographically approved
3. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism
Open this publication in new window or tab >>Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism
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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.

Keyword
nonhomologous end joining, single strand annealing
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-25628 (URN)10.1073/pnas.0903869106. (DOI)000268178400041 ()
Available from: 2008-11-25 Created: 2008-11-17 Last updated: 2012-10-31Bibliographically approved

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