Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site.
Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004257:DNA Polymerase II / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011730:Pyridoxal / Q000494:pharmacology; D011731:Pyridoxal Kinase / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D051381:Rats
D000345:Affinity Labels; D015636:Magnesium Chloride; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D011731:Pyridoxal Kinase; D004257:DNA Polymerase II; D008274:Magnesium