DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery |
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Authors: | Witte Gregor Urbanke Claus Curth Ute |
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Affiliation: | Medizinische Hochschule, Zentrale Einrichtung für Biophysikalisch-Biochemische Verfahren, Carl Neuberg Strasse 1, D-30625 Hannover, Germany. |
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Abstract: | Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the χ subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between χ and an EcoSSB/ssDNA complex when compared to that of χ to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of χ, showing that χ interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that χ specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to χ at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III. |
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