Selected precipitation of proteins from cytosol of mammalian cells with polyethylene glycol (PEG) at high ionic strength allows a rapid and efficient separation of DNA polymerases-o and $3 in a single operation which can be conducted on a large scale. With this technique, 90% of the DNA polymerase-cY is recovered in the PEG precipitate, whereas the DNA polymerase-P is exclusively recovered in the supernatant.
Mammalian
cells have been found to contain two distinct DNAdependent DNA polymerases. One of these, DNA polymerase-a has a high molecular weight (100,000 to 200,000) and is generally found in the cytosol (1,2). DNA polymerase-P has a low molecular weight (about 45,000) and is found in the nucleus and also in the cytosol (1,2). Consequently, for many studies, it will be necessary to distinguish first between DNA polymerase-a and DNA polymerase-P activities in a crude extract, and, secondly, to separate the two enzymes rapidly. It is possible to distinguish the two activities using centrifugations on sucrose gradients (3,4) and chromatography on DNA cellulose (6), DEAE-cellulose (7), or phosphocellulose (8). The distinction between the two polymerases can be achieved by studying their template properties or sensitivity to N-ethylmaleimide (l-5). All these procedures involve a lot of operations and require a long time.
This communication discusses the use of selective precipitation of DNA polymerase-cY with polyethylene glycol (PEG) at high ionic strength as a tool to separate DNA polymerase-a and DNA polymerase-P rapidly.
Methods
Preparation of cytosof extract. The liver cytosol from 12 male WAG rats, partially hepatectomized, was prepared by using a method described previously (5). Regenerating livers (65 g) removed 40 hr after hepatectomy were disrupted with a Potter homogenizer in 3 vol of 50 mM Tris-HCl (pH 7.5), 6 mM KCl, 1 mM MgC&, 1 mM 2-mercaptoethanol and centrifuged for 2 hr at 105,OOOg.