Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymic DNA synthesis
- 6 February 1990
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 29 (5), 1200-1207
- https://doi.org/10.1021/bi00457a016
Abstract
The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase.beta.-like, P. polycephalum DNA polymerase .alpha., and human placenta DNA polymerase .alpha.. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3''-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.This publication has 5 references indexed in Scilit:
- PROKARYOTIC AND EUKARYOTIC DNA-POLYMERASES .1. THE ROLE OF INTERNUCLEOTIDE PHOSPHATES IN THE BINDING PROCESS OF OLIGONUCLEOTIDE PRIMER WITH THE ENZYME1987
- Non‐disruptive detection of DNA polymerases in nondenaturing polyacrylamide gelsEuropean Journal of Biochemistry, 1985
- Replication of M13mp7 single‐stranded DNA in vitro by the 9‐S DNA polymerase α from calf thymusEuropean Journal of Biochemistry, 1984
- Enzymological characterization of KB cell DNA polymerase-alpha. III. The polymerization reaction with single-stranded DNA.Journal of Biological Chemistry, 1979
- A new method for sequencing DNA.Proceedings of the National Academy of Sciences, 1977