Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.
The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002482:Cellulose; D002846:Chromatography, Affinity; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification*; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000032:analysis* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D013604:T-Phages / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology
D004800:Enzymes, Immobilized; D000249:Adenosine Monophosphate; C029427:adenosine monophosphate-sepharose; D002482:Cellulose; D012685:Sepharose; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V