Kinetic characteristics which distinguish two forms of calf thymus DNA polymerase alpha

Biochemistry. 1981 Jan 6;20(1):227-32. doi: 10.1021/bi00504a038.


DNA polymerase alpha was isolated as previously described [Holmes, A. M., Hesslewood, I.P., & Johnston, I. R. (1974) Eur. J. Biochem. 43, 487]. This method yields five nuclease-free forms of alpha-polymerase, A1, A2, B, C, and D. Holmes and co-workers [Holmes, A. M., Hesslewood, I.P., Wickremasinghe, R. G., & Johnston, I.R. (1977) Biochem. Soc. Symp. 42, 17] have suggested that the C form is the core enzyme of alpha-polymerase and have demonstrated that removal of a protein subunit from the A1 form yields an enzyme with the physical properties of the C form. They did not investigate the function of the subunit because the A1 and C forms were not easily distinguished with biochemical kinetics. We have been able to demonstrate three kinetic differences between these forms: (1) the alpha-A1-polymerase adds more nucleotides per binding event to activated DNA (is more processive) than does alpha-C-polymerase. (2) The synthetic activity of the alpha-A1-polymerase is greater on a template with an average gap size of 65 nucleotides than it is on a template with an average gap size of 10 nucleotides whereas that of the alpha-C-polymerase is not. (3) The synthetic activity of the alpha-C-polymerase is inhibited by high concentrations of activated calf thymus DNA (greater than 300 muM) whereas that of the alpha-A1-polymerase is not. The nature of the inhibitor was investigated and found to be a nuclear RNA component present in the DNA preparations. These kinetic differences may provide a means to assay for the protein subunit that converts alpha-C-polymerase to alpha-A1-polymerase, and provide a basis for isolation and characterization of other DNA replication-association proteins.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cattle
  • DNA / biosynthesis
  • DNA Polymerase I / metabolism*
  • DNA, Single-Stranded / biosynthesis
  • DNA-Directed DNA Polymerase / metabolism*
  • Isoenzymes / metabolism*
  • Kinetics
  • Plasmids
  • Ribonucleases
  • Substrate Specificity
  • Templates, Genetic
  • Thymus Gland / enzymology*


  • DNA, Single-Stranded
  • Isoenzymes
  • DNA
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase
  • Ribonucleases