Purification and properties of a specific primase-stimulating factor of bovine thymus.
The DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.
Itaya
A
A
Department of Biochemistry, Nara Medical University, Japan.
Hironaka
T
T
Tanaka
Y
Y
Yoshihara
K
K
Kamiya
T
T
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D011506:Proteins / Q000302:isolation & purification*; D013950:Thymus Gland / Q000201:enzymology*
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011506:Proteins; C056412:primase-stimulating factor; D004259:DNA-Directed DNA Polymerase