Functional Domains of Escherichia coli Single-Stranded DNA Binding Protein As Assessed by Analyses of the Deletion Mutants

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• 作者：Takashi Kinebuchi ; Heisaburo Shindo ; Hiroki Nagai ; Nobuo Shimamoto ; and Mitsuhiro Shimizu
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：June 3, 1997
• 年：1997
• 卷：36
• 期：22
• 页码：6732 - 6738
• 全文大小：223K
• 年卷期：v.36,no.22(June 3, 1997)
• ISSN：1520-4995

文摘

A series of C- and N-terminal deletion mutants ofEscherichia coli single-stranded DNA bindingprotein (SSB) was constructed, purified, and characterized in terms ofability to self-multimerize and tobind to DNA. High-performance gel filtration chromatographyrevealed that the amino acids 89-105play a key role in the maintenance of homotetramer for native SSB of177 amino acids. Interestingly, allof the N-terminal deletion mutants studied here were eluted asoctamers, indicating that the N-terminal11 residues are involved in the prevention of the formation ofoctamers. The binding of SSB and itsdeletion mutant proteins to single-strandedd(T)_n was examined by gel mobility shift assayand circulardichroism spectroscopy. C-terminal deletion mutant proteins,SSB1-135 and SSB1-115, maintainedhigh affinity and may be wrapped by single-stranded DNA (ssDNA) in thesame way as in the case ofnative SSB. In contrast, deletion of the C-terminal region(residues 89-115) or N-terminal region (residues1-11) caused a dramatic decrease in the binding affinity.Furthermore, two different stoichiometries ofSSB in the complexes with d(T)₆₄, but not withd(T)₃₂, were observed for native SSB, SSB1-135,SSB1-115, and SSB37-177, suggesting that the (SSB)₆₅ and(SSB)₃₅ binding modes, as previouslydemonstrated[Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260,3594-3603; Bujalowski, W., & Lohman,T. M. (1986) Biochemistry 25, 7799-7802], occurred atlower and higher SSB concentrations, respectively.A functional map for SSB molecule was presented anddiscussed.