NMR Investigation of Primer-Template Models:  Structural Effect of Sequence Downstream of a Thymine Template on Mutagenesis in DNA Replication^†

Misaligned structures can occur in primer-templates during DNA replication, which can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, we have investigated how the nucleotide downstream of a thymine template affects the primer-template structures upon misincorporation of dNTPs. The base pair structures at the replicating sites of a set of primer-template models containing either a G or an A downstream of the thymine template have been determined using NMR spectroscopy. Incorporation of dCTP and dTTP opposite 5‘-GT and 5‘-AT templates, respectively, can result in misaligned structures with a T-bulge. Depending on the downstream sequence, subsequent extension of the primers may stabilize the misaligned structures or cause the formation of mismatched structures. These results provide alternative pathways for base substitution and deletion errors during DNA replication by low-fidelity polymerases.