Mutagenic potential of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass catalyzed by human Y-family DNA polymerases

One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPolη), iota (hPolι), kappa (hPolκ), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPolη, hRev1, hPolκ, and hPolι to bypass 50% of the 8-oxodG lesions encountered (t₅₀^bypass) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPolη bypassed 8-oxodG most efficiently, hPolη correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPolι bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPolκ was most accurate opposite 8-oxodG (92.3%), whereas hPolκ alone was the least accurate (18.5%). The t₅₀^bypass value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPolη alone, suggesting that hPolη outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.