To asses the possible roles of the two active forms of mouse
DNA polymerase alpha: primase--
DNA-polymerase alpha complex (
DNA replicase) and
DNA polymerase alpha free from
primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum
starvation, or Ehrlich culture cells, arrested at the S phase by
aphidicolin, showed
DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in
DNA replicase activity most likely resulted from synthesis of a new
enzyme, as shown by experiments using a specific
monoclonal antibody,
aphidicolin and
cycloheximide. Not only with respect to the presence or absence of
primase activity, but in other points as well the catalytic properties of these two forms were found to differ;
DNA replicase preferred the activated
calf thymus DNA with wide gaps of about 100
nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50
nucleotides long. Size analysis of the products synthesized on M13 single-stranded
circular DNA with a single 17-nucleotide primer by
DNA replicase and 7.3S polymerase suggested the ability of
DNA replicase to overcome a secondary structure formed in
single-stranded DNA to be greater than that of 7.3S polymerase.