Naslov
Single-stranded DNA binding protein has a key role in chromosome segregation during morphological differentiation of Streptomyces coelicolor

Autori
Filić, Želimira ; Paradžik, Tina ; Crnolatac, Ivo ; Bielen, Ana ; Piantanida, Ivo ; Herron, Paul ; Vujaklija, Dušica

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

Izvornik
Molecules of Life / Kos, Janko ; Poklar-Urlich, Nataša - Ljubljana : Slovenian Biochemical Society, 2015, 56-56

ISBN
978-961-93879-0-0

Skup
FEBS3+ Meeting

Mjesto i datum
Portorož, Slovenija, 16.09.2015. - 19.09.2015

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
SSB proteins, Gene expression, SsbA and SsbB, Crystal structures, oxidative stress
(SSB proteins, Gene expression, SsbA and SsbB, Crystal structures, Oxidative stress)

Sažetak
SSB proteins bind ssDNA with a high affinity and in a sequence independent manner thus protecting transiently formed ssDNA during DNA replication, recombination and repair. In addition, SSBs interact and modulate the activities of various proteins involved in DNA metabolism. Analysis of available bacterial genomes revealed the presence of multiple copies of SSB proteins in many bacteria. The number of ssb genes can vary even among closely related species thus indicating that evolution of these proteins in Eubacteria is highly dynamic. However, the role of duplicated SSB proteins is poorly studied. Multicellular bacterium Streptomyces coelicolor with two ssb genes was selected to study biological role(s) of paralogous SSB proteins. Gene expression analyses suggested that SsbA and SsbB may have different biological roles. In concert with this, the EMSA assays and fluorescent titrations showed that SsbA and SsbB bind to ssDNA with different affinity. In addition, results of the gene disruptions have strongly indicated that ssbA is essential for survival while ssbB is important during the sporulation process. Crystal structures of these proteins also revealed some structural variations that led us to hypothesize that SsbB binding activity might be regulated during oxidative stress in S. coelicolor. To get a better insight into the function of SsbB we examined the impact of disulfide bridges removal on SsbB activity/stability. Cysteine residue (Cys7) in SsbB was substituted by alanine or serine. The gene ssbB carrying cys7 mutation was not able to complement S. coelicolor strain lacking ssbB. Next, the study of the wild type protein and two SsbB mutants by differential scanning calorimetry showed decrease of thermal stability in the series: SsbBwt>SsbB/Ala>SsbB/Ser. Furthermore, binding of 45dT studied by isothermal titration calorimetry has strongly indicated that the concentration of the active conformation of the protein also declines in that series (SsbBwt>SsbB/Ala>SsbB/Ser). To shed the light on the complex mechanism of cell division in Streptomyces, we have constructed double (ssbBparB ; ssbBsmc) and triple (ssbBparBsmc) mutant strains carrying mutations in ssbB gene and in the genes previously reported to be important for the chromosome segregation. By fluorescence microscopy we examined the effect of these mutations. The results showed more severe defects in nucleoid segregations during sporulation than previously reported for parental strains.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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