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Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis
Abstract
Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism such as DNAreplication, repair, and recombination, and is essential for cell survival. This study reports on the ssb-likegene cloning, gene expression and characterization of a single-stranded DNA-binding protein of Pseudoal-teromonas haloplanktis (PhaSSB) and is the first report of such a protein from psychrophilic microorganism.PhaSSB possesses a high sequence similarity to Escherichia coli SSB (48% identity and 57% similarity) andhas the longest amino acid sequence (244 amino acid residues) of all the known bacterial SSBs with one OB-fold per monomer. An analysis of purified PhaSSB by means of chemical cross-linking experiments,sedimentation analysis and size exclusion chromatography revealed a stable tetramer in solution. UsingEMSA, we characterized the stoichiometry of PhaSSB complexed with a series of ssDNA homopolymers,and the size of the binding site was determined as being approximately 35 nucleotides long. In fluores-cence titrations, the occluded site size of PhaSSB on poly(dT) is 34 nucleotides per tetramer under low-saltconditions (2 mM NaCl), but increases to 54–64 nucleotides at higher-salt conditions (100–300 mM NaCl).This suggests that PhaSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB. The binding properties of PhaSSB investigated using SPR technology revealed that the affinity of PhaSSB to ssDNA is typical of SSB proteins. The only difference in the binding mode of PhaSSB to ssDNA is a faster association phase, when compared to EcoSSB, though compensated by faster dissociation rate.When analyzed by differential scanning calorimetry (DSC), the melting temperature (Tm) was determinedas 63◦C, which is only a few degrees lower than for EcoSSB.
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Authors (4)
-
Anna Cyranka-Czaja
- University of Wroclaw Faculty of Biotechnology, Department of Protein Engineering
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Full text
- Publication version
- Accepted or Published Version
- DOI:
- Digital Object Identifier (open in new tab) 10.1016/j.micres.2013.07.010
- License
- Copyright (2013 Elsevier GmbH)
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- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
-
MICROBIOLOGICAL RESEARCH
no. 169,
edition 2-3,
pages 139 - 147,
ISSN: 0944-5013 - Language:
- English
- Publication year:
- 2014
- Bibliographic description:
- Olszewski M., Nowak M., Cyranka-Czaja A., Kur J.: Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis// MICROBIOLOGICAL RESEARCH. -Vol. 169, iss. 2-3 (2014), s.139-147
- DOI:
- Digital Object Identifier (open in new tab) 10.1016/j.micres.2013.07.010
- Verified by:
- Gdańsk University of Technology
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