DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity

• Published in: The Journal of biological chemistry 1988-06, Vol.263 (18), p.8981-8988
• Main Authors: Nasheuer, H P, Grosse, F
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cattle; Chromatography, Affinity; Chromatography, Ion Exchange; DNA Primase; Fundamental and applied biological sciences. Psychology; Kinetics; Macromolecular Substances; Molecular and cellular biology; Molecular genetics; Molecular Weight; Osmolar Concentration; Replication; RNA Nucleotidyltransferases - isolation & purification; RNA Nucleotidyltransferases - metabolism; Templates, Genetic; Thymus Gland - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)68404-2

Immunoaffinity-purified DNA polymerase alpha-primase complex from calf thymus consists of subunits with molecular weights of 148,000-180,000, 73,000, 59,000, and 48,000 (Nasheuer, H.-P., and Grosse, F. (1987) Biochemistry 26, 8458-8466). Primase activity was separated from the immobilized complex by washing extensively with 2 M KCl or, alternatively, by shifting to pH 11.5 in the presence of 1 M KCl. From both elution procedures, the primase activity was found to be associated with the polypeptides with molecular weights of 59,000 and 48,000. The specific activity, using either elution procedure, was 30,000 units/mg. Both polypeptides sedimented together at 5.7 S upon zonal centrifugation on a sucrose gradient. Primase activity was found in the flow-through fraction after DEAE-cellulose chromatography of the free primase. Analysis of this fraction by sodium dodecyl sulfate gel electrophoresis revealed only one band with a Mr of 48,000. Polyclonal antibodies were raised against the Mr 59,000 and 48,000 polypeptides. The anti-Mr 59,000 antibody affected the primase activity only marginally, whereas the anti-Mr 48,000 antibody inhibited the primase activity nearly completely. UV cross-linking of the DNA polymerase alpha-primase complex with alpha-32P-labeled GTP revealed a binding site at the Mr 48,000 polypeptide, but none at the other subunits of the complex. Taken together, these results suggest that the Mr 48,000 polypeptide bears the active site of the DNA primase activity. The Mr 59,000 polypeptide stabilizes the primase activity.