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

Hockensmith JW, Bambara RA
Biochemistry (1981), Volume 20, Page 227
PubMed entry


DNA polymerase alpha was isolated as previously described [Holmes, A. ...
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.




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