Deciphering the Role of DNA Polymerase Eta on the Incorporation and Bypass of Inosine and Cell Cycle Arrest

Inosine is a key intermediate in many cellular pathways, and our RT-qPCR data showed that DNA polymerase eta (polη) was upregulated upon the repeated treatment of inosine and inosine monophosphate (IMP) in HCT116 cells, which suggests that polη is actively involved in the incorporation and bypass of inosine in cells. To gain novel insight into mutagenic potential of inosine incorporation into DNA and its implication on cell cycle arrest, we conducted structural, biochemical, and cell biological studies of human polη on the incorporation and bypass of inosine. Our nucleotide insertion assay showed that polη incorporated inosine triphosphate (ITP) opposite dC just 18-fold more efficiently than opposite dT, indicating that ITP incorporation by polη is promutagenic. Our three polη crystal structures showed that ITP formed Watson-Crick base pair with dC and that ITP adopted both syn- and anti-conformations across dT, increasing the promutagenicity. Our flow cytometry data showed that only excessive treatment of inosine and IMP caused S- and G2-phase arrest, suggesting that polη's lesion bypass activity might resolve the cell cycle arrest. Our results give us novel insights into the role of polη in the mutagenic incorporation and bypass of DNA lesions, which might affect cell cycle arrest.