Novel insights into the role of translesion synthesis polymerase in DNA incorporation and bypass of 5-fluorouracil in colorectal cancer.

5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 µM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2'-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.

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

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 (polη). However, the structural and biochemical basis for the mutagenic potential of the incorporation of deoxyinosine triphosphate (dITP) into DNA remains poorly understood. To gain insights into the mutagenic potential of the incorporation of inosine into DNA, we conducted structural and kinetic studies of human polη incorporating dITP across undamaged DNA template containing dC or dT. Polη incorporated dITP opposite dC 14-fold more efficiently than opposite dT, indicating that dITP incorporation by polη can be mutagenic unlike the bypass of inosine by polη, which incorporated dCTP almost exclusively opposite the templating inosine over dTTP (70:1). Polη-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 polη-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 incorporation by polη (190:1 across dC and dT).