Mammalian DNA polymerase α: a replication competent holoenzyme form from calf thymus

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Abstract
A complex “replication competent” holoenzyme form of DNA polymerase α (RC-α) was purified 10′000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-α form could partially replicate a double-stranded o1igo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-α was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-α required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-α with the neutralizing anti-DNA polymerase a monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386–8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-α preparation following removal of specific polypeptides strongly suggested that the capacity of RC-α to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.