High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds

Abstract

We report studies testing the importance of Watson-Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson-Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

Figure 1

(a) Structures of nucleoside shape mimics dF and dQ, which lack Watson–Crick hydrogen-bonding ability but possess shapes very similar to dT and dA (shown for comparison), respectively. (b) Space-filling models of nucleobase analogs F and Q with comparison to T and A, showing shapes with electrostatic potentials mapped on the surfaces (red, negative potential; blue, positive).

Figure 2

Replication-bypass efficiencies in E. coli for phage templates containing the F and Q isosteres, with comparison to a chemically stable tetrahydrofuran abasic analog (AP) site. The solid bars show data for normal replication, and crosshatched bars show results under UV light-induced SOS damage-response conditions from individual genomes constructed in triplicate.

Figure 3

Mutagenesis data for nonpolar DNA base isosteres in E. coli under normal (SOS-uninduced) conditions. (a) Chromatogram from the REAP assay using genomes that were constructed in triplicate with oligonucleotides containing G, Q, or F at the type IIs (BbsI) cleavage site. These genomes were passaged through E. coli that were not induced for the SOS response. The lanes designated “M” contain markers generated from an oligonucleotide with a degenerate 5′ end that was carried through the assay at the ³²P-labeling step. (b) Plots of mutagenesis data (from a) showing high fidelity of replication. A log₁₀ plot was used to make visible all insertion events.

Figure 4

DNA base isostere mutagenesis data in SOS-induced E. coli. (a) Chromatogram from the REAP assay using G-, Q-, and F-containing genomes constructed in triplicate and passaged through E. coli that were induced for the SOS response with UV light. (b) Graphical representation of mutagenesis data from G, Q, and F genomes passaged through E. coli that were induced for the SOS response with UV light (from a). The log₁₀ plot shows that the isosteres provide for direct coding when SOS replication proteins are expressed. The graph illustrates the near lack of ambiguous pairing of the isosteres with an incipient dNTP during DNA replication in vivo even with low-fidelity lesion-bypass polymerases.