Biochemistry zyxwvu 1987, 26, zyxwvu 5377-5382 5377 Scopes, R. K. (1975) Methods Enzymol. 42, 127-134. Scopes, R. K. (1978a) Eur. J. Biochem. 85, 503-516. Scopes, R. K. (1978b) Eur. J. Biochem. 91, 119-129. Sinha, N. D., Biernat, J., McManus, J., zyxwvutsr & Koster, H. (1984) Smith, M., & Gillam, S. (1981) Genet. Eng. 3, 1-32. Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A,, Perkins, R. E., Conroy, Nucleic Acids Res. zyxwvutsrqp 12, 4539-4557. S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., & Kingsman, S. M. (1982) EMBO J. 1, 1635-1640. Wrobel, J. A., & Stinson, R. A. (1978) Bur. J. Biochem. 85, Zagursky, R. J., Baumeister, K., Lomax, N., & Berman, M. Zoller, M., & Smith, M. (1983) Methods Enzymol. 101, 345-350. (1985) Gene Anal. Tech. 2, 89-94. 468-500. Yeast DNA Primase Is Encoded by a 59-Kilodalton Polypeptide: Purification and Immunochemical Characterization? Esther E. Biswas, Philomena E. Joseph, and Subhasis B. Biswas* Department of Biological Chemistry, University zyxwvut of Maryland zyxwvut School of Medicine, Baltimore, Maryland 21 201 Received February 9, 1987 ABSTRACT: The DNA primase from the yeast Saccharomyces cerevisiae has been purified 9200-fold to homogeneity. The yeast DNA primase is a monomeric protein of molecular weight 59000, and under conditions described in this report, it is stable at 4 or -80 OC. The primase does not bind to DEAE-cellulose, is not inhibited by a high concentration of a-amanitin (4 mg/mL), and is capable of synthesizing small (up to 15 nucleotides in length) rib0 or ribo-deoxy mixed initiator RNA primers. The primer synthesis is stimulated by ATP; however, other ribonucleotides could be replaced by deoxynucleotides without any measurable effect on the overall DNA synthesis. Thus, the purified primase is distinct from the RNA polymerases of S. cereuisiae. Immunoblot analysis of the polypeptides in a crude cell extract using a mouse polyclonal antibody prepared against the highly purified primase indicates that the 59-kilodalton polypeptide is the native form and not a degraded form of a larger polypeptide; however, primase is degraded rapidly to smaller polypeptides by yeast proteases especially in the absence of protease inhibitors. x e DNA primase activity has been shown to be essential for the synthesis of primers that enable DNA polymerases to initiate synthesis of Okazaki fragments in the lagging strand of the replication fork (Kornberg, 1980; Oertel & Goulian, 1977). The primase activity was demonstrated in Escherichia coli (E. coli)‘ to be distinct from the RNA polymerase, and the purified primase has been shown to be a product of the DnaG gene of E. coli (Bouche et al., 1975; Zechel et al., 1975; Rowen & Kornberg, 1978). Recent studies in several labo- ratories (Conaway & Lehman, 1982a,b; Tseng & Ahlem, 1982; Yagura et al., 1983) demonstrated that in eukaryotes, the DNA primase activity is associated at least in part with the DNA polymerase a. Vishwanatha and Baril(l986) have demonstrated recently that HeLa cell DNA primase is a 70- kDa polypeptide and could be separated from primase-po- lymerase complex by hydrophobic chromatography. In the yeast Saccharomyces cerevisiae, a large fraction of the cellular primase activity remains free (Singh & Dumas, 1984; Wilson & Sugino, 1985; Jazwinski & Edelman, 1985). However, attempts toward purification of this free primase activity have led to ambiguities with respect to its mass, specific activity, subunit structure, and physical properties (Jazwinski & Edelman, 1985; Wilson & Sugino, 1985; Plevani et al., 1985). As primase is a major component of the chromosomal apparatus, purification and characterization of primase are This work was supported in part by the Frank G. Rizer Memorial Grant for New Investigators from the American Cancer Society, Ma- ryland Division, Inc. * Address correspondence to this author. 0006-2960/87/0426-5377$01.50/0 important in deciphering the mechanism of eukaryotic chro- mosomal DNA replication. The present study has been di- rected toward developing an appropriate purification protocol for primase, establishing its subunit structure and size, and preparing a polyclonal antibody against primase for immu- nological characterization and molecular cloning of its gene. MATERIALS AND zyxwvu METHODS Yeast. S. cerevisiae (wild-type bakers’ yeast) was obtained as a gift from the American Yeast Corp., Baltimore, MD, and was removed from the fermenter during mid log phase. Nucleotides, Enzymes, and DNA. All ribo- and deoxy- nucleotides were obtained from ICN. All radioactive nu- cleotides were obtained from New England Nuclear. Sin- gle-stranded M 13 phage DNA was prepared according to the published procedure (Ray, 1969). Calf thymus DNA, yeast tRNA, and a-amanitin were obtained from Sigma Chemical Co. Calf thymus DNA was activated with DNase I according to the procedure of Fansler and Loeb (1 974). Poly(dT), de- ’ Abbreviations: E. coli, Escherichia coli; zyxw S. cerevisiae, Saccharo- myces cereuisiae; PMSF, phenylmethanesulfonyl fluoride; pepA, pep- statin A; DTT, dithiothreitol; Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(P- aminoethyl ether)-N,N,N’,N’-tetraacetic acid; ssDNA, single-stranded DNA; Me2S0, dimethyl sulfoxide; BSA, bovine serum albumin; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; Bis, N,N‘-methylene- bis(acry1amide); kDa, kilodalton(s); pol I, polymerase I; NEM, N- ethylmaleimide. 0 1987 American Chemical Society