STUDIES ON THE SUBUNIT STRUCTURE AND MOLECULAR FORM OF E. COLI DNA POLYMERASE III
SUGRUE, STANLEY JOHN
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Procedures for the isolation and purification of Escherichia coli DNA polymerase III (Pol III) from both wild-type and temperature-sensitive polC strains are described. The procedure for purification of Pol III from a polC('+) strain (HMS83) yields an enzyme preparation which exhibits a single protein band in native polyacrylamide gel electrophoresis. Denaturing gels of the most purified preparatins reveal polypeptides of 140,000, 25,000 and 10,000 daltons. The enzyme has associated with it two distinct exonuclease activities; an exonuclease which degrades single-stranded DNA in a 3' (--->) 5' direction but which does not attack duplex structure and another activity which initiates hydrolysis on single-strands with a 5' (--->) 3' specificity but which will then proceed into duplex regions effecting complete hydrolysis of a double-stranded DNA molecule having single-stranded 5'-termini. Both nuclease activities copurify with the DNA polymerase III activity and are recovered in the same yields as polymerase following physical separation of the 140,000 dalton polypeptide from the 25,000 and 10,000 dalton polypeptides by denaturing gel filtration. In addition, the 5'-exonuclease activity is found to be temperature-sensitive when isolated from a polC('ts) strain by a rapid purification procedure developed for that purpose. Physical characterization of the enzyme indicates a Stokes radius of 50(ANGSTROM) and a sedimentation coefficient of 7.0 S; the latter two parameters are consistent with a native molecular weight of 140,000 daltons for DNA polymerase III.;Evidence that the two smaller polypeptides observed in pure preparations of the enzyme are breakdown products of the 140,000 dalton subunit is described. Data on the existence of different complexes of Pol III with other DNA replication proteins is also presented as well as observations on an unusual effect of phosphate on the enzyme during gel filtration.;DNA elongation factor I was purified to homogeneity by a rapid purification procedure which is outlined. The most purified fraction exhibits a single band in native gel electrophoresis and a single polypeptide of 42,000 daltons in denaturing gels. Physical measurements indicate a Stokes radius of 30(ANGSTROM) and a sedimentation coefficient of 3.8 S, consistent with a protein having a single polypeptide chain of 42,000 daltons.