Drosophila DNA polymerase delta. Purification and characterization.
A DNA polymerase with properties similar to mammalian polymerase delta has been isolated to near homogeneity from early embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that this enzyme has a total molecular mass of 185 kDa and is composed of 138- and 47-kDa polypeptides. Its isoelectric point is 6.8. This polymerase activity is strongly inhibited by N-ethylmaleimide, aphidicolin, and high KCl concentration but is relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate. There was no reaction in an immunological test using monoclonal antibody against Drosophila DNA polymerase alpha. In a final purification step, this polymerase activity was accompanied by 3'-->5'-exonuclease activity as expected proof-reading activity. This polymerase activity is remarkably stimulated by mouse proliferating cell nuclear antigen, which is structurally and immunologically very similar to a Drosophila counterpart. These properties clearly indicate this enzyme belongs to the category of DNA polymerase delta.
Aoyagi
N
N
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Chiba-ken, Japan.
Matsuoka
S
S
Furunobu
A
A
Matsukage
A
A
Sakaguchi
K
K
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001483:Base Sequence; D002850:Chromatography, Gel; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D054306:Dideoxynucleotides; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D011189:Potassium Chloride / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen; D013942:Thymine Nucleotides / Q000494:pharmacology
D054306:Dideoxynucleotides; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D013942:Thymine Nucleotides; D016590:Aphidicolin; D011189:Potassium Chloride; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate; D005033:Ethylmaleimide