A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity

Abstract

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

Figure 1

The nucleotide structures and DNA sequences in this study. (A) Chemical structures of nucleosides T and analog F alongside space-filling models of the “nucleobases” of the two. (B) Sequences of template–primer duplexes used in steady-state studies. The standing-start substrate utilizes a 23-nt primer, whereas the running-start substrate utilizes an 18-nt primer.

Figure 2

Autoradiogram showing a survey of standing-start single-nucleotide insertions by the KF (exo⁻) enzyme, including all possible cases of analog F in the template and as a dNTP. The data were taken using 2 μM dNTP, and the reactions were stopped after 2 min.

Figure 3

Autoradiogram showing running-start primer elongations up to and beyond adenine with comparison with the known pause opposite an abasic nucleotide (φ). Reactions were performed with 20 μM of each dNTP; for the template containing A, the dNTP mixture was dATP + dCTP + dGTP + dFTP, and for the abasic template, dATP + dCTP + dGTP + dTTP.

Figure 4

Histogram showing accuracy of template selection for nucleotide insertion by the KF enzyme. Semilog plot for insertion efficiency of dFTP and dTTP opposite each of the four natural bases in a template.