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.