Kinetic characteristics which distinguish two forms of calf thymus DNA polymerase alpha.
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.
D000818:Animals; D002417:Cattle; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D012260:Ribonucleases; D013379:Substrate Specificity; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
D004277:DNA, Single-Stranded; D007527:Isoenzymes; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D012260:Ribonucleases