Logo of plntphysLink to Publisher's site
Plant Physiol. May 1994; 105(1): 69–79.
PMCID: PMC159330

Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).


DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants.

Full Text

The Full Text of this article is available as a PDF (2.9M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Badaracco G, Bianchi M, Valsasnini P, Magni G, Plevani P. Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex. EMBO J. 1985 May;4(5):1313–1317. [PMC free article] [PubMed]
  • Bartholomew B, Meares CF, Dahmus ME. Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA. J Biol Chem. 1990 Mar 5;265(7):3731–3737. [PubMed]
  • Biswas SB, Kornberg A. Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study. J Biol Chem. 1984 Jun 25;259(12):7990–7993. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Budd ME, Campbell JL. DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):496–505. [PMC free article] [PubMed]
  • Castroviejo M, Tharaud D, Tarrago-Litvak L, Litvak S. Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria. Biochem J. 1979 Jul 1;181(1):183–191. [PMC free article] [PubMed]
  • Chang LM, Rafter E, Augl C, Bollum FJ. Purification of a DNA polymerase-DNA primase complex from calf thymus glands. J Biol Chem. 1984 Dec 10;259(23):14679–14687. [PubMed]
  • Cleghon V, Klessig DF. Characterization of the nucleic acid binding region of adenovirus DNA binding protein by partial proteolysis and photochemical cross-linking. J Biol Chem. 1992 Sep 5;267(25):17872–17881. [PubMed]
  • Insdorf NF, Bogenhagen DF. DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure. J Biol Chem. 1989 Dec 25;264(36):21491–21497. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Laquel P, Castroviejo M, Litvak S. Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A. Nucleic Acids Res. 1990 Aug 25;18(16):4867–4876. [PMC free article] [PubMed]
  • Laquel P, Litvak S, Castroviejo M. Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases. Plant Physiol. 1993 May;102(1):107–114. [PMC free article] [PubMed]
  • Richard MC, Litvak S, Castroviejo M. DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase. Arch Biochem Biophys. 1991 May 15;287(1):141–150. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Santocanale C, Foiani M, Lucchini G, Plevani P. The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis. J Biol Chem. 1993 Jan 15;268(2):1343–1348. [PubMed]
  • Siegal G, Turchi JJ, Jessee CB, Mallaber LM, Bambara RA, Myers TW. Structural relationships between two forms of DNA polymerase epsilon from calf thymus. J Biol Chem. 1992 Feb 25;267(6):3991–3999. [PubMed]
  • Singh H, Dumas LB. A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae. J Biol Chem. 1984 Jun 25;259(12):7936–7940. [PubMed]
  • Wang TS, Hu SZ, Korn D. DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha. J Biol Chem. 1984 Feb 10;259(3):1854–1865. [PubMed]
  • Wang Z, Wu X, Friedberg EC. DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1051–1058. [PMC free article] [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...