Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.
A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(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 previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.
Kinebuchi
T
T
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
Shindo
H
H
Nagai
H
H
Shimamoto
N
N
Shimizu
M
M
D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D017353:Gene Deletion; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments