Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties

D. M. Livingston, D. C. Hinkle, C. C. Richardson

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31 Scopus citations

Abstract

DNA polymerase III was purified 4,500 fold from the E. coli mutant, HMS83, which lacks DNA polymerases I and II. When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered. Polyacrylamide gel electrophoresis under denaturing conditions produces 2 protein bands with molecular weights of 140,000 and 40,000. The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A. Taken together these 2 parameters indicate a native molecular weight of 180,000. Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand. The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand. Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single stranded regions. The most purified enzyme preparation is devoid of endonuclease activities. In addition to the 2 exonuclease activities described, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates. DNA polymerase III was also isolated from wild type E. coli containing the other 2 known DNA polymerases. Furthermore, the enzyme purified from 3 different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III.

Original languageEnglish (US)
Pages (from-to)461-469
Number of pages9
JournalJournal of Biological Chemistry
Volume250
Issue number2
StatePublished - 1975

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