Average Gap Size Research Articles

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.

DNA synthesis by isolated mitochondria V. Electron microscopy of replicative intermediates

1. We have analysed by electron microscopy mtDNA preparations extracted before and after incubation of isolated chick-liver mitochondria with DNA precursors. The only detectable effect of incubation was on molecules with single-stranded gaps co-sedimenting with the open circular form of mtDNA; the average gap size had decreased by 30 % after incubation. 2. Up to 90 % of classifiable molecules of the closed circular duplex fraction of rat-liver mtDNA have displacement loops. mtDNA extracted directly from chick-liver homogenates or from mitochondria isolated in the presence of ethidium bromide, an inhibitor of mtDNA synthesis, contained displacement loops of the same size and at the same frequency as mtDNA from isolated mitochondria. This indicates that D-loops are present in vivo and not formed during the isolation of the mitochondria. 3. These results and our previous biochemical experiments indicate that isolated mitochondria are capable of carrying out at least the first and last stage of the replication scheme proposed for mtDNA by Robberson D. L., Kasamatsu, H. and Vinograd, J. (1972) Proc. Natl. Acad. Sci. U.S. 69, 737–741.