The Use of Non‐natural Nucleotides to Probe Template‐Independent DNA Synthesis

Abstract

The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the "blunt-end" of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive pi-electron surface areas are efficiently utilized by the T4 DNA polymerase. These analogues possess high binding affinities that are remarkably similar to those measured during incorporation opposite an abasic site. In contrast, the k(pol) values are significantly lower during blunt-end extension when compared to incorporation opposite an abasic site. These kinetic differences suggest that the single-stranded region of the DNA template plays an important role during polymerization through stacking interactions with downstream bases, interactions with key amino acid residues, or both. In addition, we demonstrate that terminal deoxynucleotide transferase, a template-independent enzyme, can efficiently incorporate many of these non-natural nucleotides. However, that this unique polymerase cannot extend large, bulky non-natural nucleotides suggests that elongation is limited by steric constraints imposed by structural features present within the polymerase. Regardless, the kinetic data obtained from using either DNA polymerase indicate that template-independent synthesis can occur without the contributions of hydrogen-bonding interactions and suggest that pi-electron interactions play an important role in polymerization efficiency when templating information is not present.

Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

“…It is possible that such properties might lead to unproductive conformations or associations with the enzyme. Previous experiments with other unnatural nucleotides with this enzyme have also shown truncated products to be common (56,58). …”

“…Only a few studies have used TdT to incorporate unnatural nucleotides not tethered to fluorophores. In two studies, the non-natural nucleotides incorporated were indole derivatives in which fluorescence was not described (56,57), and in the other study, we documented the incorporation of 3–4 fluorescent pyrene nucleotide analogs (58) which stalled strongly at that point. The current studies are distinct from these precedents not only because of the changes in fluorescence of the incorporated dxA and dxC analogs, but also because of the strong abilities of xDNA to hybridize to natural DNA, which has previously been documented (59).…”

Quantifying the energetic contributions of desolvation and π-electron density during translesion DNA synthesis

“… a Assays were performed using 500 nM gp43 exo − , 250 nM DNA substrates and variable concentrations of non-natural nucleotide in the presence of 10 mM Mg 2+ .Kinetic values were taken from the following references: b (57), c (27), d (22), e (42) and f (35) for comparison. g ND: not determined as incorporation was too inefficient to measure accurately.…”

“…Kinetic values were taken from the following references: b (57), c (27), d (22), e (42) and f (35) for comparison.…”

“…More significant differences are observed during blunt-end extension, another form of non-instructional DNA lesion (42). As indicated in Table 1, only 5-MeCITP is utilized during blunt-end DNA synthesis, albeit with a low catalytic efficiency of 1290 M −1 s −1 .…”