MISINCORPORATION OF NUCLEOTIDES BY CALF THYMUS DNA PRIMASE AND ELONGATION OF PRIMERS CONTAINING MULTIPLE NONCOGNATE NUCLEOTIDES BY DNA-POLYMERASE-ALPHA

Misincorporation of nucleotides by calf thymus DNA primase was examine d using synthetic DNA templates of defined sequence. Primase seldom mi sincorporated NTPs during initiation of a new primer (i.e. polymerizat ion of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accur ate nucleotide-polymerizing enzyme known. In some eases primase discri minated against incorrect NTPs by less than a factor of 100. After pri mase incorporated a noncognate nucleotide into the primer, the next co rrect NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing mu ltiple mismatches. Generation of a correctly base-paired primer-templa te negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After pri mase synthesized a primer containing multiple mismatches, the primer w as transferred to the polymerase cu active site via an intramolecular mechanism. Importantly, polymerase cu readily elongated this primer if dNTPs were present. These data are discussed with respect to the ques tion of why primase is required for DNA replication.