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

Abstract

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.

Effect of hyperoxidized guanine on DNA primer–template structures: Spiroiminodihydantoin leads to strand slippage

“…2C) indicate the formation of a C17 · G4 Watson–Crick base pair, confirming the misaligned structures with a Sp‐bulge and a closing C · G Watson–Crick base pair. This structural finding is similar to our previous observations on misaligned structures with a T‐bulge [17,18] or C‐bulge [19] in pyrimidine templates.…”

“…Despite the presence of lesions can slow or stall the action of polymerase, low fidelity DNA polymerases have shown the capability to accommodate misaligned primer–template in their loose active sites [15,16], and thus, DNA replication can continue and deletion error can occur. Recently, we have demonstrated that misalignment can occur at the replicating sites of primer–templates [17–19], yet the structural effect of Sp and oG lesions at these sites remains elusive.…”

NMR proton chemical shift prediction of T·T mismatches in B-DNA duplexes

“…DNA mismatches can occur in vivo due to the following reasons: (i) misincorporation of bases [24] or strand misalignment [25][26][27][28][29] during replication, (ii) heteroduplex formation during homologous recombination [30], and (iii) spontaneous deamination [31] and damage by mutagenic chemicals or ionizing radiation [32][33][34][35][36]. Results from various research groups have shown that the mismatch repair efficiency depends on the type of mismatches and base composition of neighboring sequences [37][38][39].…”

NMR proton chemical shift prediction of C·C mismatches in B-DNA

“…Structural studies of DNA mismatches become more and more important as they have been found to occur in the double-helical stem regions of the unusual secondary structure formed by highly mutagenic and disease-related sequences [39][40][41]. These mismatches can occur in vivo during DNA replication [42][43][44][45][46][47], recombination [48], or as a result of DNA damage [49][50][51][52][53]. If left unrepaired, mismatched sites are highly mutagenic [54].…”