The Steric Gate of DNA Polymerase ι Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity

Background: Accurate bypass of DNA damage by translesion DNA polymerases is critical for cell survival. Results: Wild-type human DNA polymerase incorporates ribonucleotides, and a steric gate mutant increases both ribonucleotide incorporation and deoxyribonucleotide selectivity. Conclusion: A single amino acid residue in DNA polymerase limits incorporation of ribonucleotides into DNA. Significance: DNA polymerase may incorporate ribonucleotides during translesion DNA synthesis. Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase (pol ), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol to incorporate NTPs during DNA synthesis. pol incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A steric gate pol mutant is considerably more active in the presence of Mn2+ compared with Mg2+ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol incorporates NTPs in a template-specific manner, certain DNA sequences may be at risk for elevated mutagenesis during pol -dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.