Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface |
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Authors: | Kun-Wei Chan Chia-Hung Wang Yuh-Ju Sun |
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Affiliation: | 1 Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC 2 Institute of Biotechnology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC |
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Abstract: | Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 107 M− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)35] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues. |
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Keywords: | SSB, single-stranded DNA-binding protein OB-fold, oligonucleotide/oligosaccharide binding fold ssDNA, single-stranded DNA HpSSB, Helicobacter pylori SSB HpSSBc, C-terminally truncated HpSSB EcoSSB, Escherichia coli SSB EMSA, electrophoretic mobility shift assay |
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