Evidence that DNA Polymerase‐a of Calf Thymus Contains a Subunit of Molecular Weight 155000

Abstract

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.

Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

“…Polyacrylamide gel electrophoresis in sodium dodecyl sulphate was performed by a standard method [18] and in non-denaturing conditions as previously described [19].…”

“…A main band at a position corresponding to a molecular weight of 135000 was found together with a minor one at M , 68000. Since non-denaturing electrophoresis can result in removal of considerable amounts of protein impurity [19], the band at M , 135 000 may represent enzyme protein (compare M , from calculated molar mass). Finally, preincubation o f enzyme D with 2.8 M urea for urea for 30 min did not alter either its sedimentation coefficient or its elution position on DEAEcellulose [13].…”

“…zyme D this species sedimented at about 6.8-6.9 S but it was not further studied. For comparison, samples of A1 and C were also further purified from the first DEAE-cellulose column as described for enzyme D [6,19] omitting however, the second run on Sepharose 6B. The final specific activities of A1 and C were 3000 and 6000 units/mg, respectively, when assayed on activated DNA; for enzyme D assayed with poly(dA) .…”

“…This corresponded to an apparent molecular weight of approximately 250000 and to a diffusion coefficient D~o ,~ of 42 pm2 sC1 [5]. When ~2 0 .~ and D~O ,~ were combined in the Svedberg equation (using va of 0.725 ml/g) the calculated molar mass was approxi- protein) was subjected to electrophoresis under nondenaturing conditions [19] it moved as a single peak of activity with a relative mobility of 0.25 (compared with 0.1 1,0.14 and 0.26 for AI, AZ and C respectively). The peaks of enzyme activity extracted from five similar non-denaturing gels were pooled to give 10 units of activity (approx.…”

Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

“…The physiological significance, if any, of this heterogeneity is not clear at this time. Johnston and coworkers (11) showed that mild treatment of a high molecular weight form (200,000-250,000 daltons) of calf thymus DNA polymerase a with 2.5 M urea converts the enzyme to a 155,000-dalton form with the release of a 50,000-to 70,000-dalton protein(s). McKune and Holmes (12) reconstituted the high molecular weight form of polymerase a from the combination of the 155,000-dalton and the 50,000-to 70,000-dalton protein (s).…”