Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97

master's thesis

• Title (english): Purification and crystallization of the protein complex SPRTN:p97
• Author: Tea Bezjak
• Mentor: Nives Ivić (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Ivica Đilović (član povjerenstva)
• Committee member: Adriana Kenđel (član povjerenstva)
• Committee member: Željka Car (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2023-02-23, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

DNA-ovisna metaloproteaza Spartan (SPRTN) održava genomski integritet sudjelujući u
popravku križnih veza DNA i proteina (engl. DNA-protein crosslink, DPC), čestog tipa
oštećenja DNA. Citotoksičnost DPC-ova pripisuje se ometanju staničnih procesa koji uključuju
DNA (replikacija, transkripcija i popravak) te predstavljaju prepreku u održavanju genomske
stabilnosti. Pokazano je da SPRTN sudjeluje u vezanju AAA+ ATP-aze p97, čija je najpoznatija
uloga odmotavanje ubikvitinitanih proteina. Rješavanje kristalne strukture kompleksa
SPRTN:p97 i njegova strukturna karakterizacija omogućila bi dublje razumijevanje uloge p97
u popravku DPC oštećenja. Stoga su humani SPRTN i p97 te njihovi skraćeni konstrukti,
prekomjerno eksprimirani i pročišćeni kromatografskim metodama. Metodom povlačenja
proteina okarakterizirana je interakcija navedenog kompleksa. Kako bi se pokušalo odrediti
stehiometriju nastalog kompleksa napravljena je elektroforeza na poliakrilamidnom gelu u
nativnim uvjetima. Kompleks proteina SPRTN i p97 kristaliziran je metodom difuzije para u
svrhu rješavanja njihove kristalne strukture. Inicijalni kristalizacijski uvjeti su detaljno
optimizirani kako bi se pripremili monokristali zadovoljavajuće veličine i kvalitete, što bi u
konačnici omogučilo rješavanje kristalne strukture kompleksa SPRTN:p97.

Abstract (english)

The DNA-dependent metalloprotease Spartan (SPRTN) functions in maintaining genomic
integrity by participating in the repair of DNA-protein crosslinks (DPCs), a common type of
DNA damage. The cytotoxicity of DPCs is attributed to the disruption of cellular processes
involving DNA (e.g. replication, transcription and repair) and poses a challenge in the
maintenance of genomic stability. It was shown that SPRTN participates in the binding of the
AAA+ ATPase p97, well known for its function in unwinding of ubiquitinated proteins. Solving
the crystal structure of the SPRTN:p97 complex and its functional characterization would allow
a deeper understanding of the p97 role in DPC damage repair. Therefore, human SPRTN and
p97, as well as several of their deletion mutants were overexpressed and purified by standard
chromatography techniques. Subsequently, complex formation between SPRTN and p97 was
tested and characterized via pull-down assays. To estimate the stoichiometry of the complex,
native polyacrylamide gel electrophoresis was applied. SPRTN and p97 complex was subjected
to crystallization by vapor diffusion method. Initial crystallization conditions were further
optimized with the aim to obtain larger and higher quality diffracting crystals. Taken together,
this set the stage for obtaining the structure of the SPRTN:p97 complex.

• Keywords: popravak DNA; unakrsna veza DNA-protein; metaloproteaza Spartan; kristalizacija proteina; pročišćavanje proteina
• Keywords (english): DNA repair; DNA-protein crosslink; Sprtn metalloprotease; protein crystallization; protein purification
• Language: croatian
• URN:NBN: urn:nbn:hr:217:838249
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-02-23
• Created on: 2023-03-01 12:05:02