Ekspresija domene SprT proteina ACRC i analiza vezanja ACRC i DNA in vitro

master's thesis

• Title (english): Expression of ACRC SprT domain and ACRC:DNA binding analysis in vitro
• Author: Josipa Valičević
• Mentor: Nives Ivić (mentor)
• Committee member: Dubravka Matković-Čalogović (predsjednik povjerenstva)
• Committee member: Jasmina Rokov Plavec (član povjerenstva)
• Committee member: Đani Škalamera (član povjerenstva)
• Committee member: Marko Močibob (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2021-09-29, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Unakrsna veza DNA-protein (engl. DNA-protein crosslink, DPC) specifičan je tip oštećenja
molekule DNA koji nastaje kao posljedica ireverzibilne kovalentne veze proteina i DNA. S
obzirom na važnost genetičke informacije pohranjene u DNA, oštećenja kao što su DPC
rezultirala bi gubitkom integriteta molekule DNA. Proučavanjem metaloproteaza, Wss1 i
SPRTN, otkriveno je da sadrže domenu SprT koja sudjeluje u popravku DPC-a. Nedavno je
otkriveno da protein ACRC (engl. Acidic containing-repeat protein, ACRC) također ima
očuvanu domenu SprT, što daje zaključiti da je uključen u popravak spomenutog oštećenja.
Kako bi se strukturno i biokemijski okarakterizirala domena SprT proteina ACRC dizajnizani
su konstrukti domene različitih duljina. Konstrukti su klonirani, testirana je njihova
nadekspresija u bakteriji Escherichia coli i njihova topljivost. U želji za razumijevanjem
uloge ACRC-a u popravku DPC-a pročišćeni su humani proteini ACRC i njegov katalitički
mutant E593A, te je ispitano njihovo vezanje na različite molekule DNA (dvolančanu,
jednolančanu i u obliku rašlji), a od sva tri tipa najbolje je vezanje na DNA u obliku rašlji.

Abstract (english)

DNA-protein crosslink (DPC) is a specific type of DNA damage that occurs as a result of the
formation of an irreversible covalent bond between DNA and protein. Given the importance
of genetic information stored in DNA, damage such as DPC would result in losing DNA
integrity. Previously, metalloproteases Wss1 and SPRTN were found to contain the SprT
domain involved in DPC repair. Recently, it has been discovered that ACRC (Acidic
containing-repeat protein) protein also has the preserved SprT domain, which suggests its
involvement in DPC repair. In order to structurally and biochemically characterize the SprT
domain of the ACRC protein, domain constructs of different lengths were designed. The
constructs were cloned, their overexpression in Escherichia coli and their solubility were
tested. In order to understand the role of ACRC in DPC repair, human ACRC protein and its
catalytic mutant E593A were purified and their binding to different DNA molecules (doublestranded, forked and single-stranded) were examined. Of all three types, binding to the fork
type of DNA molecule was the best.

• Keywords: :ACRC; izolacija proteina; unakrsna veza DNA-protein; vezanje i popravak DNA
• Keywords (english): ACRC; DNA binding and repair; DNA-protein crosslink; protein isolation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:485031
• Project: Number: IPS-2020-01-4225; Title: Strukturna karakterizacija faktora uključenih u popravak unakrsnog vezanja DNA-protein; Leader: Marta Popović; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IPS
• 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: Open access; Embargo expiration date: 2023-09-29
• Created on: 2021-10-26 13:37:12