The high-molecular-weight DNA polymerase fraction of calf thymus, rat liver or rat spleen can be resolved into further activities differing from one another in molecular weight and charge properties. The use of Sepharose 6B columns brings about partial resolution of these activities although DEAE-cellulose achieves a more effective separation. The high-molecular-weight fraction from calf thymus has so far yielded four species of activity, A, B, C and D sedimenting at 7.9 S, 5.2 S, 7.3 S and 6.0 S, respectively. In rat liver and spleen, 7.6-S (I), 7 . 2 3 (11) and 5-S enzymes have been detected although smaller and more variable amounts of 5-S enzyme occur in the rat tissues compared with calf thymus where the 5.2-5 species can represent 20-
Small samples of the 8‐S species of enzymes (A1 and A2) occurring in the DNA polymerase‐a fraction of calf thymus, have been extensively purified using non‐denaturing (normal) polyacrylamide gel electrophoresis. When peak fractions of activity on normal gels were subjected to dodecylsulphate‐polyacrylamide gel electrophoresis, a polypeptide at 155000 correlated with polymerase activity. Samples of the 7.3‐S (C) enzyme prepared from A2 by treatment with 2.4 M urea or isolated directly without exposure to urea, also showed the presence of a 155000‐Mr polypeptide. It is concluded that the 7.3‐S (C) enzyme, of previously estimated molecular weight 155000–170000, is a single polypeptide and that the 8‐S enzymes A1 and A2 contain an additional subunit of 50000–70000 molecular weight.
The heterogeneity of calf thymus DNA polymerase-a has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)lo (A : T = 20 : 1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-a fraction (enzymes A1 and C) which do not show a preference for poly (dA) . (dT)lo over activated DNA.As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-a in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly(A) . (dT)Io template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-p. The extreme sensitivity of enzyme D to inhibition by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 "C unlike enzyme Al.The possible interrelationships of the multiple activities of calf thymus DNA polymerase-a are discussed.Several different types of DNA polymerase activity have been described in mammalian cells [1,2]. Of these DNA polymerase-a is of particular interest since its activity is highest in situations where DNA synthesis is occurring [3]. A further point of interest concerns the heterogeneity of DNA polymerase-a reported in several instances, for example in calf thymus [4-71 rat liver and spleen [5], Chinese hamster cells [8], mouse myeloma [9-111 and baby hamster kidney (BHK) cells [12]. When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]). However, assay of the same DEAEcellulose profile with the synthetic template-initiator complex poly(dA) . (dT)lo at pH 7.0 reveals a further
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