Identification of a novel protein, PriB, in Klebsiella pneumoniae |
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Authors: | Hsieh Hui-Chuan Huang Cheng-Yang |
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Affiliation: | aDepartment of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan;bDepartment of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan |
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Abstract: | PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L45, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed. |
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Keywords: | Abbreviations: ssDNA, single-stranded DNA SSB, single-stranded DNA-binding protein EMSA, electrophoretic mobility shift analysis nt, nucleotides aa, amino acids Kd, the apparent dissociation constant OB, oligonucleotide/oligosaccharide binding EcSSB, E. coli ssDNA-binding protein |
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