The role of DNA polymerase η in UV mutational spectra

Abstract

UV irradiation generates predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine–thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase η (Pol η) dependent process. Pol η is encoded by the POLH ( XPV) gene in humans. In order to clarify the specific role of Pol η in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells. This strategy provides an advantage over studying mutagenesis in cell lines derived from normal individuals and XP-V patients, since the genetic background of the cells is identical. Synthetic RNA duplexes were used to inhibit Pol η expression in 293T cells. The reduction of Pol η mRNA and protein was greater than 90%. The supF shuttle vector was irradiated with UVC and replicated in 293T cells in presence of anti-Pol η siRNA. The supF mutant frequency was increased by up to 3.6-fold in the siRNA knockdown cells relative to control cells confirming that Pol η plays an important role in mutation avoidance and that the pol η knockdown was efficient. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Surprisingly, neither the type of mutations nor their distribution along the supF gene were substantially different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. The data are compatible with two models. (i) Incorrect replication of cytosine-containing photoproducts by a polymerase other than Pol η produces similar mutations as when Pol η is present but at a higher frequency. (ii) Due to lack of Pol η or low levels of remaining Pol η, lesion replication is delayed allowing more time for cytosine deamination within CPDs to occur. We provide proof of principle that siRNA technology can be used to dissect the in vivo roles of lesion bypass DNA polymerases in DNA damage-induced mutagenesis.