Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii.
A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.
Wang
Z F
ZF
Department of Biological Sciences, University of Maryland Baltimore County 21228.
Yang
J
J
Nie
Z Q
ZQ
Wu
M
M
D000645:Ammonium Sulfate; D002696:Chlamydomonas / Q000201:enzymology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000645:Ammonium Sulfate