Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

Biochemistry. 1991 Jan 29;30(4):1127-31. doi: 10.1021/bi00218a034.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ammonium Sulfate
  • Chlamydomonas / enzymology*
  • Chromatography, Affinity
  • Chromatography, DEAE-Cellulose
  • DNA / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Electrophoresis, Polyacrylamide Gel
  • Plasmids

Substances

  • DNA
  • DNA-Directed DNA Polymerase
  • Ammonium Sulfate