Isolation and characterization of DNA polymerases from mature oocytes of the starfish, Asterina pectinifera.
Four distinctive DNA polymerases have been isolated from mature oocytes of the starfish, Asterina pectinifera, and characterized. The N-ethylmaleimide-resistant DNA polymerase activity was separated from the other three DNA polymerases by hydroxyapatite column chromatography. The enzyme, whose approximate sedimentation coefficient was 3.3S, effectively utilized poly(dA)-oligo(dT) as a template-primer and was completely inhibited by 10 microM 2', 3'-dideoxythymidine-5'-triphosphate but was not inhibited by aphidicolin. Therefore, this enzyme was classified as a eukaryotic DNA polymerase-beta. Three species of the N-ethylmaleimide-sensitive DNA polymerase were separated with a phosphocellulose column. Two (5.5S and 6.5S) of the enzymes eluted from the column preferred activated DNA as a template-primer. Both of the DNA polymerases were inhibited by aphidicolin but not by 2',3'-dideoxythymidine-5'-triphosphate. Judging from the above results, these two DNA polymerases were classified as eukaryotic DNA polymerase-alpha. The third N-ethylmaleimide-sensitive enzyme (approximately 7S) eluted from the phosphocellulose column utilized poly(rA)-oligo(dT) as well as poly(dA)-oligo(dT) as template-primers. The enzyme activity was resistant to aphidicolin but sensitive to 2',3'-dideoxythymidine-5'-triphosphate. This enzyme resembles eukaryotic DNA polymerase-gamma. Thus, it is concluded that the four species of DNA polymerase found in starfish oocytes resemble eukaryotic DNA polymerase-alpha, -beta, and -gamma.
D000818:Animals; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004259:DNA-Directed DNA Polymerase / Q000145:classification / Q000302:isolation & purification*; D005260:Female; D007525:Isoelectric Focusing; D009865:Oocytes / Q000201:enzymology*; D010063:Ovum / Q000201:enzymology*; D013215:Starfish / Q000201:enzymology*; D013698:Templates, Genetic
D004259:DNA-Directed DNA Polymerase