Interaction of drosophila DNA polymerase α holoenzyme with synthetic template-primers containing mismatched primer bases or propanodeoxyguanosine adducts at various positions in template and primer regions

We studied recognition and binding of synthetic template-primers by Drosophila DNA polymerase α (pol α) holoenzyme. The template-primers used contained either mismatched base pairs at various positions in the primer region or exocyclic propanodeoxyguanosine (PdG) adducts at various positions in both template and primer. pol α requires primer-terminal complementarity of greater than or equal to 4 base pairs for efficient binding and incorporation. When a mismatched base pair is at the -4 position relative to the 3′-primer terminus, minimal but detectable binding occurs. This is consistent with the ability of pol α to incorporate a single nucleotide on a template-primer containing a mismatch at this position, but at a rate of only 7% relative to incorporation on a perfectly matched template-primer. No binding or incorporation (less than 1% of incorporation on a perfectly matched template-primer) was evident when a mismatched base pair was at the -3 position or closer, relative to the 3′-primer terminus. Similar results were obtained when PdG was placed at various positions in the primer region. When a PdG residue was located in the template region (+3 position relative to the 3′-primer terminus), single-nucleotide incorporation was stimulated 3-4-fold. These observations suggest that there are intrinsic aspects to the mechanism of nucleotide incorporation by pol α which ensure the fidelity of DNA synthesis by this enzyme and may provide novel insights into the fundamental mechanism of polymerase translocation along templates.