An investigation of potential multi-enzyme complexes of DNA precursor synthesis and DNA replication in eukaryotic cells
1. Efforts to display a 'replitase' complex in two disparate lower eukaryotes, Saccharomyces cerevisiae and Physarum polycephalum whether employing physical or kinetic techniques have yielded no evidence to support its existence at this level of biological complexity. 2. Some indication of potential interaction of the folate-metabolising enzymes, dihydrofolate reductase and thymidylate synthase, were attained from affinity chromatography and non-denaturing gel electrophoresis studies of S. cerevisiae lysates. 3. Experiments on lysates prepared from S. cerevisiae spheroplasts imply a cytoplasmic location for the enzymes NDP kinase, dihydrofolate reductase, thymidylate kinase and thymidylate synthase while DNA polymerase, by virtue of its much reduced activity in such extracts, appears to be non-cytoplasmic. 4. Lysates of S-phase P. polycephalum macroplasmodia and exponentially-growing microplasmodia have different DNA polymerase elution profiles when subjected to Sepharose 6B gel filtration chromatography implying the existence of an S-phase-specific activity. 5. Fractions from the trailing section of the S-phase-specific peak of P. polycephalum DNA polymerase following gel filtration chromatography are capable of utilising [3H]dTMP but not [3H]TdR as substrate. The substrates of dTMP synthetase (dUMP plus co-factors) are not capable of substituting for dTTP in the DNA polymerase assay in these fractions. 6. Thymidylate synthase does not seem to be physically linked with the DNA polymerase from S-phase BHK-21/C13 cells.