Solubilization and partial characterization of a multienzyme complex of DNA synthesis from human lymphoblastoid cells.
1. Gently lysed human lymphoblastoid cells (HPB-ALL) catalysed incorporation of the distal DNA precursors [3H]thymidine, [3H]dTMP, and [3H]dUMP into DNA. Measurement of the [3H]dTTP, produced during the incorporation reactions, provided kinetic evidence for the channelling of the distal precursors into DNA by a multienzyme complex consisting of precursor-synthesizing enzymes and of DNA polymerase. 2. In the presence of the DNA polymerase inhibitor, 1-beta-D-arabinofuranosylcytosine triphosphate, incorporation of [3H]dTMP into DNA was abolished. However, there was no accumulation of [3H]dTTP, suggesting that the flow of substrates through the complex was regulated by the activity of DNA polymerase. 3. Multienzyme complexes were detected by chromatography of a lysate on a column of Sepharose 6B. These complexes retained the ability to channel [3H]thymidine and [3H]dTMP into DNA.
D002460:Cell Line; D002850:Chromatography, Gel; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D012995:Solubility; D013698:Templates, Genetic
D004247:DNA; D004259:DNA-Directed DNA Polymerase