Affinity Chromatography of DNA‐Binding Enzymes on Single‐Stranded DNA‐Agarose Columns

Abstract

Agarose gels containing immobilized single-stranded circular DNA from phage fd or denatured calf thymus DNA were investigated for their use in the affinity chromatography of DNA-binding enzymes. The DNA content of gel fragments is stable under the conventional conditions of enzyme purification. Single-stranded DNA-agarose columns have a high capacity to bind DNA-specific proteins. They were used to differentiate between similar enzymatic activities in DNA-free extracts from Escherichia coli. Preparative purification is described for the following enzymes : E. coli DNA polymerase I, DNA polymerase 11, RNA polymerase, exonuclease I11 and T, polynucleotide kinase. Enzyme purification was as high as 200-fold, recovery of enzymatic activity was 75-10Oo/,.Affinity chromatography [l], which is based on the specific binding of enzymes to their substrates or substrate analogs fixed t o an insoluble matrix, was first applied in the field of nucleic acid research by Alberts et al. [2] and Litman [3], who introduced DNA-cellulose as a chromatographic resin. This material has been used extensively for the detection and purification of DNA-specific proteins from various organisms [4--81. Trapping of DNA in polyacrylamide-agarose gels and chemical coupling to agarose beads [9,10] has also been used to immobilize DNA for affinity chromatography. I n all these studies double-stranded DNA was preferred as a ligand to imitate physiological conditions.We has a high binding capacity for many DNA-specific enzymes. It can be used very effectively in the analytical and preparative affinity chromatography of these proteins. MATERIAL AND METHODSE . coli H 560 endl-, polA-, kindly provided by Dr. H. Hoffmann-Berling, was grown in a minimal medium. The growth and harvesting of the cells, in late log phase, was carried out by Merck AG (Darmstadt). E. coli W cells were a gift of Farbenfabriken Bayer AG (Leverkusen). Cells were frozen and stored at -20 "C until used.Calf thymus DNA type V was purchased from Sigma (St. Louis, U.S.A.), fd DNA was prepared as described [ 131. Agarose (for electrophoresis) was purchased from Serva (Heidelberg) or Sigma (St. Louis, U.S.A). Whatman DEAE-cellulose DE23 was precycled and equilibrated as described by the manufacturer. Sarkosyl NL 97 was a gift from Geigy (Basel).Standard buffer was 0.01 M Tris pH 7.5, 0.1 mM dithioerythritol, 5 o/o (v/v) glycerol. Varying amounts of NaCl or KC1 were added as indicated. The dithioerythritol was added to the buffer just prior to use. The ionic strength of solutions was determined by measuring their conductivity and comparing it with that of standard solutions of NaCI. It is given in the equivalent NaCl concentration. DNA concentration and DNA polymerase I1 [21]. RNAase activity was measured by degradation of 32P-labelled RNA and is given in arbitrary units. Preparution of DNA-AguroseThe procedure described by Bendich and Bolton [12] was used except that the DNA was dissolved in dilute alkali to reduce the viscosity of the highly concentrated DNA solution. Calf thymus DN...

A DNA primase specified by I-like plasmids.

“…Agarose SeaKem HGT(P) was from Marine Colloids, Rockland, ME. DNA-agarose was prepared as described (21).…”

Yeast RNA‐polymerase B: A zinc protein

“…All buffers used (except for the first purification steps) were metal freed by chromatography (without added EDTA and mercaptoethanol) on chelating resin from 100-300 nM down to less than 10 nM. DNA-agarose was prepared according to [18]. Baker's yeast was from Lindenmayer & Co., Heidelberg or Sinner KG, Karlsruhe.…”

The Study of DNA . RNA-Polymerase Complexes by Kinetic Formaldehyde Method

“…The enzyme was further chromatographed on DNA-agarose, which was treated according to Schaller et al [16]. To store the purified enzyme it was concentrated with the use of small DEAEcellulose columns (1 ml DE-52/10 mg RNA polymerase).…”