Form Of DNA Polymerase Alpha Research Articles

HeLa cell DNA polymerase alpha is tightly associated with tryptophanyl-tRNA synthetase and diadenosine 5',5"'-P1,P4-tetraphosphate binding activities.

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.

Variation of two forms of DNA polymerase-alpha during a HeLa cell cycle.

HeLa cells contain two forms of DNA polymerase-alpha (P-I and P-II) with varied affinity to DNA, separable on a DNA-cellulose column. The activity of each form was followed during a cell cycle of synchronized culture of the HeLa cells. P-I was recovered from the nuclear extract and P-II from the cytoplasmic fraction. The P-I activity remained at a low level during M to G1 phase until a marked increase between late G1 and S phases, while P-II activity increased gradually throughout the period. Both activities attained their highest level at mid-S phase and then the P-I activity declined more rapidly than the P-II. Addition of hydroxyurea at mid-S phase inhibited the decrease of both enzyme activities. The enzyme activity of nuclear extract from S phase cells was not inhibited by mixing with extract from M phase cells. When the cytoplasmic fraction from M phase cells was chromatographed on a DNA-cellulose column, single activity peak was observed at the position of P-II. These results suggest that the decrease in P-I activity is neither due to the presence of an inhibitor nor to mere release of the enzyme from chromosomes.

Separation of two forms of HeLa DNA polymerase alpha with different binding affinity to DNA.

Two forms of DNA polymerase alpha were separated from HeLa cells by DEAE-cellulose column chromatography and affinity chromatography using a DNA-cellulose column. They were eluted from a DEAE-cellulose column at 0.22M KCl (P-I) and 0.24M KCl (P-II). Upon cell fractionation between cytosol and nuclei, P-I was recovered from nuclei and P-II from cytosol. P-I was found to have a higher binding affinity to DNA than P-II by DNA-cellulose column chromatography.