This chapter describes the assay method of DNA polymerase II from Escherichia coli. The assay of DNA polymerase II from Escherichia coli is based upon the incorporation of radioactively labeled deoxynucleotide residues into acid-insoluble polydeoxynucleotides. The assay is dependent upon added DNA. The chapter describes the isolation and properties of the enzyme. Purified DNA polymerase II requires all four deoxynueleoside 5-triphosphates, template DNA, and MgCI2 for extensive synthesis of DNA. The activity is stimulated by β-mercaptoethanol or dithiothreitol, and may be eliminated by sulfhydryl-blocking agents, such as N-ethylmaleimide. The purified DNA polymerase II has a specific activity of 100–200 units/mg of protein, representing 1–2 gmoles of total nucleotide incorporation into acid-insoluble form per milligram of protein in 30 minutes at 37°.
ASJC Scopus subject areas
- Molecular Biology