Enhancing the “A-Rule” of Translesion DNA Synthesis:  Promutagenic DNA Synthesis Using Modified Nucleoside Triphosphates

Abstract

Abasic sites are mutagenic DNA lesions formed as a consequence of inappropriate modifications to the functional groups present on purines and pyrimidines. In this paper we quantify the ability of the high-fidelity bacteriophage T4 DNA polymerase to incorporate various promutagenic alkylated nucleotides opposite and beyond this class of non-instructional DNA lesions. Kinetic analyses reveal that modified nucleotides such as N6-methyl-dATP and O6-methyl-dGTP are incorporated opposite an abasic site far more effectively than their unmodified counterparts. The enhanced incorporation is caused by a 10-fold increase in kpol values that correlates with an increase in hydrophobicity as well as changes in the tautomeric form of the nucleobase to resemble adenine. These biophysical features lead to enhanced base-stacking properties that also contribute toward their ability to be easily extended when paired opposite the non-instructional DNA lesion. Surprisingly, misincorporation opposite templating DNA is not enhanced by the increased base-stacking properties of most modified purines. The dichotomy in promutagenic DNA synthesis catalyzed by a high-fidelity polymerase indicates that the dynamics for misreplicating a miscoding versus a non-instructional DNA lesion are different. The collective data set is used to propose models accounting for synergistic enhancements in mutagenesis and the potential to develop treatment-related malignancies as a consequence of utilizing DNA-damaging agents as chemotherapeutic agents.

Mechanisms of DNA Polymerases

“…124 Thus, discrimination is caused by a 1 000-fold reduction in the k pol value rather than perturbations in binding affinity. This is consistent with the induced-fit model for polymerase fidelity 29 because the rate-limiting step for nucleotide incorporation is the conformational change step preceding phosphoryl transfer as validated by thio-elemental effects studies, 124 pulse-chase experiments, 126 and fluorescence measurements of nucleotide incorporation. 127 Furthermore, extension beyond an abasic site is reduced ∼1 000-fold compared to extension beyond a correct base pair.…”

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits

“…For example, 6-Cl-2-APTP (Figure 1c), has two hydrogen bonding profiles, allowing its incorporation opposite both T and C bases. 9,15,38 Compared to dATP, 6-Cl-2-APTP replaces the 6-amino group with chlorine, but introduces a 2-amino functionality; this configuration ultimately provides the same number of Watson-Crick hydrogen bonds complementary to T as dATP. When used as a dGTP analog, 6-Cl-2-APTP has different tautomerization, which changes the N-1 from a hydrogen bond donor to an acceptor.…”

“…1–3 By forming base pairs with a shape and size similar to the canonical A•T and G•C base pairs, isosteric and hydrophobic dNTP analogs incapable of hydrogen bonding with native, complementary bases have been successfully incorporated by DNA polymerases. 4–6 Studies with other dNTP analogs substantiate a requirement for shape complementarity 7–9 and demonstrate that additional factors, including stereochemistry, sterics, electronic effects, base stacking, and hydrophobic interactions contribute to the fidelity of DNA polymerases. 8,10–15 …”

Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

“…Therefore, the bypass of abasic site by pol Á or pol Á core mainly follows a "G-rule" then the traditional "A-rule". Although a number of cellular studies show that abasic sites preferentially code for dATP insertion ("A-rule") [4], other DNA polymerases do not follow the A-rule. Human pol beta prefers to insert G opposite abasic sites [8] and S. solfataricus Dpo4 are preferred to produce -1 frameshift deletions to a greater extent than A insertion [9].…”

“…These abasic sites are very blocking and miscoding. Abasic sites preferentially code for dATP insertion (the "A-rule") [4]. For example, human pol Á was inefficient in the bypass of abasic site, and the majority of bypass events were insertions of A [5][6][7].…”