Mutagenic incorporation of inosine into DNA via T:I mismatch formation by human DNA polymerase eta

Inosine is a key intermediate in de novo purine nucleotide biosynthesis in cells. Inosine is known to be mutagenic when it is present in DNA, in place of adenine via deamination, by facilitating the incorporation of dCTP exclusively, resulting in A:T to G:C mutation. The structural basis for the mutagenicity of inosine bypass has been reported in some DNA polymerases including human DNA polymerase eta (pol eta). However, the structural and biochemical basis for the mutagenic potential of the incorporation of deoxyinosine tri-phosphate (dITP) into DNA remains poorly understood. To gain insights into the muta-genic potential of the incorporation of inosine into DNA, we conducted structural and kinetic studies of human pol eta incorporating dITP across undamaged DNA template con-taining dC or dT. Pol eta incorporated dITP opposite dC 14-fold more efficiently than oppos-ite dT, indicating that dITP incorporation by pol eta can be mutagenic unlike the bypass of inosine by pol eta, which incorporated dCTP almost exclusively opposite the templating inosine over dTTP (70:1). Pol eta-dC:dITP crystal structure showed that the incoming dITP formed Watson-Crick base pair along with wobble base pair via 4-imino-2-keto tautomer of cytosine diminishing the catalytic efficiency compared to dGTP incorporation across dC. In addition, the crystal structure of pol eta-dT:dITP revealed that dT and dITP formed Watson-Crick like base pair via 4-enol-2-keto tautomer of thymine, reinforced by wobble base pair via 4-keto-2-keto tautomer of thymine resulting in the increased mutagenicity of dITP incorporation (14:1 across dC and dT), which is 14-fold higher than dGTP incorp-oration by pol eta (190:1 across dC and dT).