Activity levels of mouse DNA polymerase alpha-primase complex (DNA replicase) and DNA polymerase alpha, free from primase activity in synchronized cells, and a comparison of their catalytic properties.
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.
D000818:Animals; D016590:Aphidicolin; D001483:Base Sequence; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D002453:Cell Cycle; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004257:DNA Polymerase II / Q000378:metabolism*; D004270:DNA, Circular / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic
D004270:DNA, Circular; D004224:Diterpenes; D016590:Aphidicolin; D003513:Cycloheximide; C023140:DNA replicase; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase