SSB與PriB結合單股DNA的模式與比較

單股DNA結合蛋白質 (SSB) 與引子合成體蛋白質PriB兩者皆為一種單股DNA結合蛋白質，作用於細菌的DNA代謝程序上。在此研究，我們藉由結構的分析、電泳遲滯分析法(EMSA)、膠體過濾色層分析法、以結構為基準的定點突變及酵母菌雙雜交法來比較這兩個蛋白質的結構功能關聯性，並得到下列結論：(1) 鼠傷寒沙門氏桿菌(St) 與克雷白氏肺炎桿菌(Kp) 的SSB為四套體，其單股DNA結合位尺寸不受高鹽與低鹽環境影響，分別為22 ± 1核苷酸 (nt) 與26 ± 1 nt。(2) KpSSB的Arg73-Ser76分子間氫鍵對於協同結合是重要的作用力之一。(3) KpPriB結合不同長度的DNA均產生一個複合體，顯示具有高度協同性。由結構與突變分析發現其Phe42與Arg44亦牽涉了與單股DNA的結合，因此DNA可完全纏繞整個PriB。(4) StPriB與不同長度DNA之結合產生多個DNA-蛋白質複合體，此結果不同於KpPriB與大腸桿菌的PriB (EcPriB)；我們推測PriB協同結合於25 nt長的DNA模式可能至少有 (EcPriB25) 與 (StPriB25) 兩種。(5) KpPriB與KpSSB C端雜合而成的KpPriB-KpSSBc蛋白質，其DNA結合性質反倒與SSB相似。根據這些不同蛋白質的結構與功能性分析，我們推測在演化上，PriB需用與不同於SSB的方式與DNA相互作用，以更有效的聚集DNA再啟動之引子合成體蛋白質群以活化停止的複製叉。

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.