Abstract Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is not well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage shows that, in the B- form, these sequences share common structural alterations which may explain the high rate of replication errors. In (CA)(n) tracts, a "Janus-like" structure with shifted base pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy which can mislead the polymerases. A model of the rat polymerase β bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base pairing while at the same time escaping enzymatic mechanisms of error discrimination scanning for the correct geometry of the minor groove. In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study reveals that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The comparison of the conformation of the nascent template-primer duplex in five available crystal structures of DNA polymerase-DNA complexes shows indeed that polymerase β the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.