Non-natural nucleotides as probes for the mechanism and fidelity of DNA polymerases

Abstract

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides.

Figure 1

(A) DNA as presented in linear, two-dimensional projections. (B) Hydrogen bonding interactions between natural nucleobases. (C) Three-dimensional representation of typical B-form DNA highlighting the influence of hydrogen-bonding interactions, steric constraints, π-π stacking interactions, and hydrophobicity on its structure.

Figure 2

(A) Structural comparison of non-natural base pairs of iso-guanine and iso-cytosine with the natural base pair guanine: cytosine. (B) Tautomerization of (C) Structural comparison of non-natural base pair of xanthine and 5-(2,4 diaminopyrimidine) with the natural base pair guanine: cytosine.

Figure 3

(A) Structures of the 2-amino-6-(N,N-dimethylamino)purine: pyridin-2-one base pair. (B) Base pairing interactions of 2-amino-(6-thienyl)purine with pyridin-2-one versus cytosine.

Figure 4

Structural comparison of non-natural base pair of adenine:2,4-difluorotoluene with the natural base pair adenine: thymine.

Figure 5

Structures of nonpolar thymidine mimics that contain various halides at positions corresponding to 2- and 4- of thymine.

Figure 6

Structures of 7-azaindole and isocarbostyril nucleosides

Figure 7

(A) Structures for benzimidazole, 4-methylbenzimidazole, 5,6-dinitrobenzimidazole. (B) Example of the “evolution” of the low fidelity non-natural nucleotide, benzimidazole, into a high-fidelity natural nucleotide. Intermediates such as 3-deazapurine and purine display incremental enhancements in fidelity for selective pairing partners.

Figure 8

Mechanism for the non-enzymatic formation of an abasic site, a non-instructional DNA lesion.

Figure 9

Structure of pyrene triphosphate that is incorporated opposite an abasic site.

Figure 10

Structural comparisons of the non-natural nucleotide, 5-NITP, with natural purine nucleotide, dATP.

Figure 11

Structures of various 5-substituted indolyl-2′-deoxyribose-5′-triphosphates used to probe the influence of π-electron stacking interactions on translesion DNA synthesis.

Figure 12

(A) Structure-activity relationship for the incorporation of various 5-substituted indolyl-deoxynucleotides opposite an abasic site. (B) Structure-activity relationship for the incorporation of various 5-substituted indolyl-deoxynucleotides opposite a templating thymine.

Figure 13

(A) Chemical structures of various pyrole and pyrazole nucleotide analogs. (B) Structures of various triazole carboxamide nucleotide analogs.

Figure 14

(A) Comparison of the base pair formed between 9-methyl-1H-imidazo-[4,5-b]pyridine (dQ) and thymine (T) versus the base pair formed between 9-methyl-1H-imidazo-[4,5-b]pyridine (dQ) and 2,4-difluorotoluene (dF). (B) Comparison of the base pair formed between 4-methylimidazole (dZ) and thymine (T) versus the base pair formed between 4-methylimidazole (dZ) and 2,4-difluorotoluene (dF). As described in the text, all four base pairs are similar with respect to shape and size. However, differences in the minor groove contacts with respect to hydrogen bonding functional groups influence the kinetics of elongation.

Figure 15

(A) Chemical structure of the self-pair formed between 7-azaindole. (B) Chemical structure of the self pair formed between 3-fluorobenzene. The natural base pair of adenine: thymine is provided as a reference.

Figure 16

Structure of the base pairing combination of dMMO2 and d5SICS.