| Summary: | 碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 100 === Single-stranded DNA (ssDNA)-binding protein (SSB) and PriB, kinds of single-stranded DNA-binding proteins, play important roles in DNA metabolism. In this study, we compared their structure-function relationships by structural analysis, electrophoretic mobility shift analysis (EMSA), gel filtration chromatographic analysis, structure-based mutational analysis, and the yeast two hybrid analyses. We found that: (1) Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) and Klebsiella pneumoniae SSB (KpSSB) respectively bound to 22 ± 1 nucleotides (nt) and 26 ± 1 nt per tetramer independent on salt concentrations. (2) An intermolecular hydrogen bond Arg73-Ser76 directed the self assembly of KpSSB on DNA.(3) KpPriB formed a single complex with ssDNA of different lengths, suggesting a highly cooperative process. Structure-based mutational analysis revealed that the known ssDNA interaction sites of PriB were expanded to include F42 and R44, allowing nucleic acids to wrap around the whole PriB. (4) Unlike KpPriB and Escherichia coli PriB (EcPriB), which form a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed. (5) The ssDNA binding properties of the chimeric protein KpPriB-KpSSBc were similar to those of KpPriB, but not KpSSB. Based on these results, as well as information on structures, we proposed that during evolution, the DNA binding strategy for PriB must be changed in order to more precisely fit the requirement for assembly of the replication re-initiation primosome at the stalled DNA forks.
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