INITIATION OF RNA-PRIMED DNA-SYNTHESIS IN-VITRO BY DNA-POLYMERASE ALPHA-PRIMASE

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequ ent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-prima se complex (pol alpha-primase), a natural DNA template containing a si te for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex D NA molecule with an open helix replication 'bubble' to serve as an ini tiation zone. Pol alpha-primase recognizes the open helix region and i nitiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ss DNA-dsDNA junction in the replication 'bubble' template is the preferr ed site for initiation. Using a 40 base oligonucleotide template conta ining the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC -3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the op en helix structure of the replication 'bubble' directs pol alpha-prima se to initiate RNA primer synthesis near the ssDNA-dsDNA junction.