Purification and properties of DNA polymerase III

Otto, B; Bonhoeffer, F; Schaller, H

doi:10.1111/j.1432-1033.1973.tb02777.x

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Purification and properties of DNA polymerase III

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Otto, B., Bonhoeffer, F., & Schaller, H. (1973). Purification and properties of DNA polymerase III. European Journal of Biochemistry, 34(3), 440-447. doi:10.1111/j.1432-1033.1973.tb02777.x.

Cite as: https://hdl.handle.net/21.11116/0000-000C-4571-7

Abstract

DNA polymerase III was purified 15000 to 20000-fold from crude extracts of Escherichia coli cells. Purification was followed by two assays, a standard polymerase assay and a complementation test in which DNA polymerase III stimulates the DNA replication in vitro of a dnaE mutant. The enzyme seems to be a single polypeptide chain with a molecular weight of about 140000. There are about 10 molecules per cell.
For maximal activity the enzyme requires all four deoxynucleoside triphosphates, Mg2+, and a high concentration of primer sites on a single-stranded DNA template. The rates of nucleotide incorporation were determined to be as high as 5000 nucleotides per min and enzyme molecule. The high sensitivity of the enzyme to salt (0.1 M KCl) is eliminated if enzyme and template are used in high concentration. In adition to its polymerizing activity the enzyme has an exonucleolytic activity on single-stranded DNA.