Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis.

Abstract

The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.