Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5'-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos.
DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.
D000345:Affinity Labels; D000818:Animals; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors*; D019915:DNA Primase; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D013698:Templates, Genetic
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V