Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.
We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D004247:DNA; D003852:Deoxyribonucleoproteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D013950:Thymus Gland / Q000032:analysis*
D000596:Amino Acids; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D004247:DNA