DEOXYRIBONUCLEIC ACID POLYMERASE ALPHA-REGULATION BY PHOSPHORYLATION

• Main Author: Vinocour, Jeanne Michelle
• Other Authors: Gerner, Eugene
• Language: en_US
• Published: The University of Arizona. 1980
• Subjects: DNA > Synthesis.; Phosphorylation.; Cellular control mechanisms.
• Online Access: http://hdl.handle.net/10150/282825

Deoxyribonucleic acid (DNA) replication in eukaryotic cells requires a highly complex series of protein-DNA interactions. Elucidation of the mechanisms by which DNA replication occurs is vital to the understanding of cellular growth. DNA polymerase alpha is an enzyme with a putative role in the replication of eukaryotic DNA. Modification by phosphorylation and dephosphorylation is one process by which enzymatic activity is regulated. The purpose of this research was to determine if a phosphorylation event could be of significance in the expression of DNA polymerase alpha activity. Evidence will be presented for the regulation of DNA polymerase alpha by phosphorylation. A highly purified DNA polymerase alpha fraction was prepared from Chinese hamster ovary cells. Purification procedure included ion-exchange chromatographies and affinity chromatography. Both the crude DNA polymerase alpha activity and the highly purified DNA polymerase alpha activity were stimulated six-fold by the addition of exogenous bovine cardiac muscle cyclic AMP-dependent protein kinase. Dephosphorylation of the highly purified DNA polymerase alpha fraction by alkaline phosphatase resulted in a concomitant decrease in DNA polymerase alpha activity. An endogenous protein kinase activity was detected in the highly purified DNA polymerase alpha fraction. Incubation of this fraction in a protein kinase reaction mixture including adenosine triphosphate (ATP) could stimulate DNA polymerase alpha activity to twelve-fold that observed in controls with no pre-incubation. The endogenous protein kinase activity in the highly purified DNA polymerase alpha fraction was utilized to indicate (1) the linear increase in DNA polymerase alpha activity with time of phosphorylation which was dependent on the presence of ATP, (2) the linear relationship between γ³²P-ATP incorporation and DNA polymerase alpha activity, and (3) the incorporation of labelled phosphate into DNA polymerase alpha as determined by SDS-PAGE analysis. Finally, the co-purification of the endogenous protein kinase with DNA polymerase alpha is presented. The significance of this research in relation to the heterogeneous nature reported for DNA polymerase alpha is discussed. It is speculated that the phosphorylational regulation of DNA polymerase alpha may play a role in the transformation of cells.