Open AccessThree Forms of DNA Polymerase from Drosophila melanogaster Embryos
Author(s) -
BRAKEL Christine L.,
BLUMENTHAL Alan B.
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12456.x
Subject(s) - enzyme , dna polymerase , microbiology and biotechnology , size exclusion chromatography , chemistry , polymerase , chromatography , sepharose , dna , primer (cosmetics) , biochemistry , gel electrophoresis , polyacrylamide gel electrophoresis , biology , organic chemistry
DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE‐cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE‐cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer‐templates, and . At the final step of purification, enzymes I, II, and III were purified approximately 1700‐fold, 2000‐fold and 1000‐fold, respectively, on the basis of their activities with The DNA polymerase eluted heterogeneously as anomalously high‐molecular‐weight molecules from Sepharose 6B gel filtration columns. On DEAE‐cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5‐7.3 S, enzyme II sedimented at 7.3‐8.3 S, and enzyme III sedimented at 7.3‐9.0 S. All enzymes were active with both synthetic primer‐templates, except the 9.0 S component of enzyme III, which was inactive with Non‐denaturing polyacrylamide gel electrophoresis did not separate activity from activity. The DNA polymerase preferred (with Mg 2+ ) as a primer‐template, although it was also active with (with Mn 2+ ), and it preferred activated calf thymus DNA to native or heat‐denatured calf thymus DNA. All three primer‐template activities were inhibited by N ‐ethylmaleimide. Enzyme activity with activated DNA and was inhibited by K + and activity with was stimulated by K + and by spermidine. The optimum pH for enzyme activity with the synthetic primer‐templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase‐α and enzymatic properties similar to those of all three vertebrate DNA polymerases.