Karakterizacija proteinskih kompleksa proteina SSB

master's thesis

• Title (english): Characterisation of SSB protein complexes
• Author: Anja Kostelac
• Mentor: Dušica Vujaklija (mentor)
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Sunčica Bosak (član povjerenstva)
• Committee member: Ana Previšić (član povjerenstva)
• Committee member: Zlatko Liber (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-02-18, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Streptomiceti predstavljaju najbrojniji rod unutar koljena Actinobacteria. Bakterija Streptomyces coelicolor A3(2) je modelni organizam za proučavanje procesa stanične diferencijacije i proizvodnje antibiotika kod bakterija. Genom ove bakterije ima dva gene, ssbA i ssbB za proteine koji vežu jednolančanu molekulu DNA (SSB), a sudjeluju u različitim procesima metabolizma DNA. Također ima i dva gena, hupA i hupS, koji kodiraju za proteine HU. Ovi proteini su združeni s nukleoidom i važni su za očuvanje strukture i integriteta DNA unutar stanice. Protein HU je potencijalni proteinski partner proteina SsbA. U ovom je radu, proteini SsbA i HupS prekomjerno su eksprimirani u heterolognom domaćinu, bakteriji Escherichia coli u svrhu pročišćavanja veće količina proteina. Za potrebe ispitivanja kompleksa SSB:HU ispitane su interakcije protein-DNA proteina SsbA i SsbB s jednolančanom DNA. Rezultati su pokazali da protein SsbB ima prosječno dva puta veći afinitet prema jlDNA nego protein SsbA, te da nastajanje kompleksa SsbB:jlDNA ne ovisi značajnije o prisutnosti soli u odnosu na kompleks SsbA:jlDNA. Pokazano je da je nastajanje kompleksa SsbA:HU spontana reakcija s visokom konstantom disocijacije i da je vođena hidrofobnim interakcijama. Interakcija između proteina SsbA i HU se ostvaruje bez prisustva jlDNA, dok se konformacijska promjena uočava samo na proteinu HU.

Abstract (english)

Streptomyes are the largest genus of Actinobacteria. Streptomyces coelicolor A3(2) is a model organism for the study of bacterial differentiation and antibiotic production. This bacterium has two genes, ssbA and ssbB, encoding single-stranded DNA binding proteins (SSBs) which participate in various processes of DNA metabolism. It also possesses two genes, hupA and hupS, encoding nucleoid-associated HU proteins which are involved in maintaining DNA integrity in the cell. In previous study we identified HU protein as a potential SSB protein partner. In this study, SsbA and HupS proteins were overexpressed in heteroulogous host, Escherichia coli and a large amount of proteins was purified for further analyses. In order to characterise complex SsbA:HU, protein-DNA interactions were analysed between proteins SsbA and SsbB and single-stranded DNA (ssDNA). The results showed protein SsbB has two-fold increased affinity with ssDNA compared to protein SsbA and that formation of SsbB:ssDNA is less salt dependent compared to SsbA:ssDNA. This study showed that formation of SsbA:HU complex is a spontaneous reaction with high dissociation constant, which is driven by hydrophobic interactions. Protein-protein interactions between SsbA and HU are formed without the presence of ssDNA, whereas conformational changes were observed only for HU protein.

• Keywords: Streptomyces coelicolor; protein SSB; protein HU; jednolančana DNA; interakcije
• Keywords (english): Streptomyces coelicolor; SSB protein; HU protein; single-stranded DNA; interactions
• Language: croatian
• URN:NBN: urn:nbn:hr:217:603412
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2021-02-18
• Created on: 2019-04-02 08:28:21