WHEAT DNA PRIMASE - RNA PRIMER SYNTHESIS IN-VITRO, STRUCTURAL STUDIESBY PHOTOCHEMICAL CROSS-LINKING, AND MODULATION OF PRIMASE ACTIVITY BYDNA-POLYMERASES

DNA primase synthesizes short RNA primers used by DNA polymerases to i nitiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subuni ts, suggesting a high degree of analogy between wheat and yeast primas e subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linkin g 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 primas e synthesizes long RNA products. The size of the RNA product synthesiz ed by wheat primase is considerably reduced by the presence of DNA pol ymerases, suggesting a modulatory effect of the association between th ese two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DN A primase using oligoadenylate [oligo(rA)]-primed or unprimed polythym idilate 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 conce ntration, Thus, Mn2+ ions led to long RNA products in a very wide rang e of concentrations, whereas with Mg2+ long products were observed aro und 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A sho wed the highest activity, followed by DNA polymerases B and CII, where as DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of th ese polymerases in DNA replication in plants.