Purification of Drosophila DNA polymerase zeta by REV1 protein-affinity chromatography.
Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.
Takeuchi
Ryo
R
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda-shi, Chiba-ken 278-8510, Japan.
Oshige
Masahiko
M
Uchida
Makiyo
M
Ishikawa
Gen
G
Takata
Kei-ichi
K
Shimanouchi
Kaori
K
Kanai
Yoshihiro
Y
Ruike
Tatsushi
T
Morioka
Hiroshi
H
Sakaguchi
Kengo
K
D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology; D004622:Embryo, Mammalian / Q000201:enzymology; D004625:Embryo, Nonmammalian; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats
D029721:Drosophila Proteins; D021122:Protein Subunits; D004247:DNA; C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase