A biophysical view on the interaction of SSB proteins with single-stranded DNA (CROSBI ID 674045)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa | međunarodna recenzija
Podaci o odgovornosti
Kostelac, Anja ; Godinić-Mikulčić, Vlatka ; Vujaklija, Dušica
engleski
A biophysical view on the interaction of SSB proteins with single-stranded DNA
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.
SSB proteins ; biophysics ; fluorescence spectrophotometry
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Podaci o prilogu
102-103.
2019.
objavljeno
Podaci o matičnoj publikaciji
Molecular Biophysics: ABC of the puzzle of life
Zagreb: Institut Ruđer Bošković ; Hrvatsko biofizičko društvo
978-953-7941-28-4
Podaci o skupu
Molecular Biophysics: ABC of the puzzle of life
poster
10.03.2019-20.03.2019
Zagreb, Hrvatska