A Potential Chemotherapeutic Strategy for the Selective Inhibition of Promutagenic DNA Synthesis by Nonnatural Nucleotides

Abstract

This manuscript reports the development of nonnatural nucleotide analogues that are preferentially incorporated opposite an abasic site, a common form of DNA damage. Competition experiments confirm that all of the nonnatural nucleotides tested are poorly incorporated into unmodified DNA. However, two analogues that contain extensive pi-electron density (5-nitro-indolyl-2'deoxyriboside triphosphate (5-NITP) and 5-phenyl-indolyl-2'deoxyriboside triphosphate (5-PhITP)) are selectively inserted opposite an abasic site and can prevent the incorporation of natural dNTPs. We demonstrate that the DNA polymerase is unable to extend beyond the incorporated nonnatural nucleotide, a result that provides direct evidence for their unique chain termination capabilities. Furthermore, these nonnatural analogues are more slowly excised once inserted opposite the DNA lesion compared to natural dNTPs. The rate of excision becomes significantly faster when the nonnatural analogues are paired opposite natural templating positions, a result that provides additional evidence for their preferential insertion opposite the DNA lesion. Moreover, idle turnover measurements confirm that the bacteriophage T4 polymerase more stably incorporates 5-NIMP and 5-PhIMP opposite damaged DNA compared to natural dNTPs. The reduced idle turnover of these analogues reflects favorable insertion kinetics coupled with reduced exonuclease-proofreading capacity. Collectively, these data demonstrate the ability to selectively inhibit translesion DNA synthesis in vitro. A novel strategy is proposed to potentially use these nucleoside analogues to enhance the chemotherapeutic effects of DNA damaging agents as well as a possible chemopreventive strategy to inhibit promutagenic DNA replication.