| The DNA primase-DNA polymerase protein complex is required for nuclear chromosomal DNA replication in the yeast S. cerevisiae. This complex is composed of four subunits, 180-, 86-, 58-, and 49-kDa. The DNA primase heterodimer (58-kDa and 49-kDa) and associated enzymatic activity have been previously studied. The catalytic DNA polymerase 180-kDa subunit and the 86-kDa subunit polypeptides have been purified using immunoaffinity chromatography to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. A four subunit reconstituted complex was detected when the 180-kDa, 86-kDa, and DNA primase heterodimer were coincubated in vitro. The reconstituted four subunit complex behaved as a single species, exhibiting a Stokes radius of 80A and a sedimentation coefficient of 8.9S. I infer from the calculated molecular weight of 312,000 that the stoichiometry is one of each subunit per complex.;While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, I have not detected a complex containing only the 86-kDa and the DNA primase heterodimer. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed. Therefore, the 86-kDa subunit was not an obligatory "linker" subunit for complex formation. However, the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex, and increased the total fraction of DNA primase activity associated with DNA polymerase activity.;The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis which had been previously observed in the DNA primase-DNA polymerase protein complex. The apparent DNA polymerase processivity was not affected by the presence of the 86-kDa subunit, but was reduced by increased Mg... |
