PLANT PHYSIOLOGY , Vol 105, Issue 1 69-79, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases)
P. Laquel, S. Litvak and M. Castroviejo
Institut de Biochimie Cellulaire, Centre National de la Recherche Scientifique, 1 rue Camille Saint Saens, 33077 Bordeaux Cedex, France
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.
