The Steric Gate of DNA Polymerase {iota} Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity [DNA and Chromosomes]

February 14th, 2014 by Donigan, K. A., McLenigan, M. P., Yang, W., Goodman, M. F., Woodgate, R.

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select deoxyribonucleotides (dNTPs) from a nucleotide pool dominated by ribonucleotides (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 iota (pol iota), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol iota to incorporate NTPs during DNA synthesis. Pol iota incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A steric gate pol iota 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 iota is able to discriminate between NTPs and dNTPs during DNA synthesis. Since wild-type pol iota incorporates NTPs in a template-specific manner, certain DNA sequences may be at-risk for elevated mutagenesis during pol iota-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol iota 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.