Enhancing the "A-rule" of translesion DNA synthesis: promutagenic DNA synthesis using modified nucleoside...

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.