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dc.date.accessioned2013-03-12T08:38:26Z
dc.date.issued2011en_US
dc.date.submitted2011-06-10en_US
dc.identifier.citationHeggelund, Julie Elisabeth. Human Endonuclease V - Cloning, Expression, Purification and Biochemical Characterization. Masteroppgave, University of Oslo, 2011en_US
dc.identifier.urihttp://hdl.handle.net/10852/11451
dc.description.abstractMultiple DNA damaging agents cause deaminations of the bases of DNA. If not repaired, these can lead to mutations, which in turn can result in dangerous diseases. Endonuclease V (EndoV) is a highly conserved gene from prokaryotes to humans. Prokaryotic EndoV has affinity for deaminated bases in DNA. Escherichia coli EndoV recognizes and binds to deaminated bases, and cleaves the DNA strand at the second phosphodiester bond, 3’ of the lesion. Structural studies of Thermotoga maritima EndoV reveals a DNA strand-separating wedge on the protein surface, containing several fully conserved residues in the EndoV family. The residues making up the DNA binding pocket, as well as the site for catalytic activity are also characterized, and are highly conserved in all EndoV homologues. Until now, structural and biochemical characterization of human EndoV, and its possible role in DNA repair, has not been the subject of any published study. The extraordinary high degree of conservation in the EndoV family suggests an important function also in the eukaryotic cell. In this thesis, we present the human EndoV (hEndoV) successfully expressed and purified for the first time. Through a series of DNA binding studies we show that human EndoV has specific DNA binding affinity for duplex DNA with distortions – like branching points, loops and flap structures. In contrast to E. coli EndoV, no binding to deaminated inosine was observed. Human EndoV was also tested for endonucleolytic activity, but no such activity has yet been detected. We also present an interaction study between human EndoV and human Slx4, to investigate a possible stimulation of branched DNA binding of hEndoV by Slx4. The results suggest no interaction. The purified protein was used to verify the binding of commercial hEndoV antibodies to our human EndoV protein. A crystal structure of human EndoV is important in order to study the protein-DNA interaction in detail, and extensive crystallization screening was performed without positive results. Additionally, small angle X-ray scattering experiments were carried out, and the resulting low-resolution structures of a DNA hairpin and a hEndoV-DNA complex are presented.eng
dc.language.isoengen_US
dc.titleHuman Endonuclease V - Cloning, Expression, Purification and Biochemical Characterizationen_US
dc.typeMaster thesisen_US
dc.date.updated2012-02-24en_US
dc.creator.authorHeggelund, Julie Elisabethen_US
dc.date.embargoenddate10000-01-01
dc.rights.termsDette dokumentet er ikke elektronisk tilgjengelig etter ønske fra forfatter. Tilgangskode/Access code Aen_US
dc.rights.termsforeveren_US
dc.subject.nsiVDP::473en_US
dc.identifier.bibliographiccitationinfo:ofi/fmt:kev:mtx:ctx&ctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft.au=Heggelund, Julie Elisabeth&rft.title=Human Endonuclease V - Cloning, Expression, Purification and Biochemical Characterization&rft.inst=University of Oslo&rft.date=2011&rft.degree=Masteroppgaveen_US
dc.identifier.urnURN:NBN:no-28898en_US
dc.type.documentMasteroppgaveen_US
dc.identifier.duo128795en_US
dc.contributor.supervisorBjørn Dalhusen_US
dc.identifier.bibsys113876866en_US
dc.rights.accessrightsclosedaccessen_US
dc.identifier.fulltextFulltext https://www.duo.uio.no/bitstream/handle/10852/11451/2/Heggelund-Master.pdf


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