DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha.
A very highly purified fraction of KB cell DNA polymerase-alpha, prepared with a monoclonal antibody, contains DNA primase activity. The primase synthesizes oligonucleotide chains initiated with ATP in a reaction that is resistant to alpha-amanitin and strictly dependent on added template and ribonucleoside triphosphates (rNTPs). In the presence of added dNTPs and M13 DNA template, the primase produces a uniform population of oligoribonucleotides, predominantly hexamers to decamers, that are extended by polymerase-alpha into DNA chains up to 3000 nucleotides long. There is no evidence for nucleotide preferences at RNA/DNA junctions. In the absence of added dNTPs, the oligomeric products are heterogeneous in size and composition and susceptible to cleavage by pancreatic DNase I due to their content of short oligodeoxynucleotide tracts synthesized by primase from trace contaminant dNTPs in the rNTP substrates. The primase and polymerase-alpha activities are distinguishable by several physical and chemical criteria, and the primase reaction is only partially sensitive to two potent, independent monoclonal antibodies that neutralize polymerase-alpha. Although the presence of both primase and polymerase-alpha activities in a highly purified immune complex prepared with a monoclonal antibody argues for their tight physical association, the chemical, physical, and immunological discriminations indicate the two catalytic entities are functionally and structurally distinct.
D002277:Carcinoma; D002460:Cell Line; D002846:Chromatography, Affinity; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009062:Mouth Neoplasms; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic
D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II