Abstract
An immunoabsorbent column, prepared by covalently linking mouse monoclonal anti-calf thymus DNA polymerase-alpha to Protein A-Sepharose, was used as the primary purification step for rapid isolation of DNA polymerase-alpha from calf thymus-gland extracts. In a 4-step procedure consisting of the removal of nucleic acids by protamine sulfate precipitation, chromatography on the immunoabsorbent column, desalting on Sephadex G-50, and removal of bovine immunoglobulins on Protein A-Sepharose, DNA polymerase-alpha activity was purified about 5000-fold from the crude extract with greater than 40% recovery of total enzyme activity. The antibody column-purified DNA polymerase-alpha fraction contains a DNA primase activity that is efficient in replication of single-stranded DNA and poly(dT) when rNTPs are included in the replication reactions. Synthesis by calf thymus DNA polymerase-primase is totally dependent on added template. Complete replication of circular single-stranded phage DNA is achieved with polymerase-primase producing a nicked circular DNA containing oligoribonucleotide primer in the final product. Primers synthesized with single-stranded phage DNA as template were up to 10 nucleotides long when dNTPs were omitted from the reaction and 8 or less nucleotides long when dNTPs were present. Primers synthesized using poly(dT) consisted of three populations when dATP was absent from the reaction, averaging 20 nucleotides, 10 nucleotides, and 3-4 nucleotides. The 20-nucleotide population was not found when dATP was included in the reaction.
Highlights
An immunoabsorbentcolumn, prepared by cova- cell enzyme [9] produced a high-molecular-weightenzyme lently linkingmouse monoclonal anti-calf thymusDNA complex, butthepurification schemeusedresulted in 1%
Ina 4- purification of DNA polymerase-a from calf thymus glands step procedure consisting of the removal of nucleic on a reusable immunoabsorbent column with excellent recovacids by protamine sulfate precipitation, chromatog- ery of enzyme activity
Primers synthesized with single-stranded phage DNA as template wereup to 10 nucleotides long when dNTPs were omitted from the reaction and 8 or less nucleotides long when dNTPs were present.Primers synthesized using poly(dTc)on
Summary
1 2 0 0K pl reaction containing 0.04 M potassium phosphate buffer at pH 7.0, 0.2 mg/ml of activated calf thymus DNA, 1 mM dithiothreitol, 8 mM. The supernatant solution obtained after centrifugation a t 8500 rpm Protein A-Sepharose treatment, the fraction was dialyzed against 25 for 30 min in a GS-3 rotor in aSorvall 5B centrifuge still showed mM potassium phosphate at pH 7.4,0.2 mM dithiothreitol, and 50%. With 250 ml of Buffer A, 50 ml of 1 M NaCl in 0.05 M Tris-HCI buffer Agarose gel electrophoresis of DNA samples was carried out on 1%. Protein remainingon the columnwas eluted with 3.2 M MgC12 DNA bands by staining with ethidium bromide, the gel wasdried buffered with 0.05 M Tris-HCI at pH 8.0. Electrophoresis was carried out ona sequencing gel apparatus at3000 V for 2.5 to 3 h
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