TY - JOUR
T1 - [2] The Isolation and Properties of DNA Polymerase II from Escherichia coli
AU - Moses, Robb E.
N1 - Funding Information:
Inzubation Procedure. The standard incubation mixture 3 contains: 66 mM Tris, pH 8.0; 6.6 mM MgC12; 1.6 mM dithiothreitol; 17 mM ammonium sulfate; 33 ~M, each, dCTP, dATP, dGTP, dTTP (one labeled with a radioactive isotope) ; 0.25 mM activated DNA; and the enzyme. The standard reaction mixture is incubated at 37 ° for 30 minutes and the reaction is terminated by the addition of 3 ml of 10% trichloroacetic acid-0.1 M sodium pyropbosphate. Enough DNA is present that it is not 1 Parts of this investigation were supported by Public Health Service Grant No. USPH GM19122-01 and American Cancer Society Grant No. ACS VC 97. R. E. Moses is a recipient of Career Development Award No. USPH GM70314-01. H. V. Aposhian and A. Kornberg, J. Biol. Chem. 237, 519 (1962). R. E. Moses and C. C. Richardson, Biochem. Biophys. Res. Commun. 41, 1557 (1970).
PY - 1974/1/1
Y1 - 1974/1/1
N2 - 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°.
AB - 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°.
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U2 - 10.1016/0076-6879(74)29004-9
DO - 10.1016/0076-6879(74)29004-9
M3 - Article
C2 - 4605284
AN - SCOPUS:0016018385
SN - 0076-6879
VL - 29
SP - 13
EP - 22
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -