Naslov
A biophysical view on the interaction of SSB proteins with single-stranded DNA

Autori
Kostelac, Anja ; Godinić-Mikulčić, Vlatka ; Vujaklija, Dušica

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Molecular Biophysics: ABC of the puzzle of life / - Zagreb : Institut Ruđer Bošković ; Hrvatsko biofizičko društvo, 2019, 102-103

ISBN
978-953-7941-28-4

Skup
Molecular Biophysics: ABC of the puzzle of life

Mjesto i datum
Zagreb, Hrvatska, 10.03.2019. - 20.03.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
SSB proteins ; biophysics ; fluorescence spectrophotometry

Sažetak
Streptomyces species are the largest genus of Actinobacteria. This genus is represented by the model organism, Streptomyces coelicolor. The replisome of S. coelicolor is composed of the following enzymes: DNA polymerase, primase, helicase, ligase, topoisomerase II, SSB and initiation proteins. SSB proteins are essential to all life forms. These proteins bind transiently single-stranded DNA (ssDNA) formed during various cellular events. SSB proteins interact with many different proteins and orchestrate DNA replication, repair and recombination processes [1]. S. coelicolor has two paralogous SSB proteins - SsbA and SsbB. SSB proteins are homotetramers that bind ssDNA in multiple binding modes ; (SSB)35 and (SSB)65 [2]. In this project, SsbA gene from S. coelicolor was cloned in expression vector, overexpressed in Escherichia coli and sequentially purified on two chromatographic columns. Binding affinities for GC-rich and poly(dT) oligonucleotides were determined by electrophoretic mobility shift assay (EMSA) for SsbA and SsbB in the presence of different NaCl concentration. The length of the shortest oligonucleotide bound by SsbA was determined by EMSA. Transition between SsbA binding modes was explored as a function of the NaCl concentration. Kinetics of photophysical intramolecular deactivation processes (Stern- Volmer relationship) of SsbA and SsbB were investigated by fluorescence spectroscopy and quenching constants were calculated. In the end, intermolecular competition between SsbA and SsbB for the same ssDNA was analysed in the presence of Mg2+ ions and different NaCl concentrations by EMSA and Western blotting. This study provides novel evidence of differential binding affinities of paralogous SSBs to ssDNA in S. coelicolor [3, 4, 5]. These results shed the new light upon assembly of SSB-DNA complexes.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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