Characterisation of the DNA endonuclease subunit EME1 as novel substrate of sumoylated Ubc9
Abstract: Sumoylation of yeast homolog of ubiquitin-conjugating enzyme Ubc9 (Ubc9), the only E2 conjugating enzyme of the sumoylation pathway, can regulate small ubiquitin-like modifier (SUMO) substrate discrimination. A screen for novel substrates of sumoylated Ubc9 identified homolog 1 of Schizosaccharomyces pombe essential meiotic endonuclease 1 (EME1) and homolog of Sacharomyces cerevisiae SLX4 structure specific endonuclease subunit (SLX4) as enhanced modified substrates of sumoylated Ubc9. This proteins are subunits of the heterodimeric DNA endonucleases MUS81-EME1 and SLX1-SLX4 and are involved in homologous recombination during mitosis. This thesis aimed to investigate the regulation of the DNA endonuclease subunit EME1 with regard to a possible regulation by sumoylation. In addition, the regulation of Ubc9 sumoylation was also investigated.
The properties of EME1 sumoylation by sumoylated Ubc9 were investigated in vitro. Important motifs of EME1 sumoylation were mapped using a mutagenic approach. The lysine 27 was demonstrated to be the major consensus site lysine for in vitro sumoylation. Additionally, a SIM motif required for sumoylation by sumoylated Ubc9 was identified. Fluorescence studies revealed an influence of the sumoylation of EME1 on its localisation. Investigation of camptothecin resistance in cells, ectopically expressing different EME1 variants, demonstrated enhanced resistance of cells expressing a sumoylation mimicking mutant of EME1. This indicates that EME1 sumoylation is required for an efficient DNA repair.
Investigation of the regulation of Ubc9 sumoylation revealed that Ubc9 sumoylation is upregulated during mitosis. This coincides with the peak activity of the tetrameric complex composed of MUS81-EME1 and SLX1-SLX4.
In summary, this thesis characterised EME1 as a novel substrate for sumoylated Ubc9 and demonstrated that EME1 sumoylation reduces the sensitivity of cells to camptothecin
- Standort
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Deutsche Nationalbibliothek Frankfurt am Main
- Umfang
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Online-Ressource
- Sprache
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Englisch
- Anmerkungen
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cc_by_nc_nd http://creativecommons.org/licenses/by-nc-nd/4.0/deed.de cc
Albert-Ludwigs-Universität Freiburg, Dissertation, 2016
- Klassifikation
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Biowissenschaften, Biologie
- Schlagwort
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Genetik
Enzym
- Ereignis
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Veröffentlichung
- (wo)
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Freiburg
- (wer)
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Universität
- (wann)
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2016
- Urheber
- DOI
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10.6094/UNIFR/11116
- URN
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urn:nbn:de:bsz:25-freidok-111166
- Rechteinformation
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- Letzte Aktualisierung
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25.03.2025, 13:46 MEZ
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