Duplex DNA binding activity of the avian myeloblastosis virus DNA polymerase

Abstract

Purified avian myeloblastosis virus DNA polymerase has a strong binding affinity for closed circular double-stranded DNA with no 3′-hydroxy termini. Because of this affinity the DNA polymerase can retain labeled native ColE1 DNA on nitrocellulose filters. When the reaction contains four enzyme molecules per ColE1 molecule about 50% of the DNA is retained. Higher enzyme: DNA ratios cause retention of nearly 100% of the DNA. The binding activity comigrates with DNA synthetic activity through ion-exchange chromatography and glycerol gradient centrifugation, an indication that it is an intrinsic activity of the DNA polymerase. Escherichia coli DNA polymerase I and bacteriophage T4 DNA polymerase do not show this binding activity, which suggests that it is not a common property of DNA polymerases. A novel application of enzyme kinetics using endonuclease-treated DNA reveals the relative quantities of enzyme molecules which are synthesizing at 3′-termini vs. molecules bound to double-stranded regions. Heat stability measurements indicate that the polymerizing activity of the enzyme can be almost completely eliminated while about half of the original binding activity is retained.