Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn²⁺ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg²⁺. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn²⁺ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn²⁺ activated the reaction a thousand-fold compared to Mg²⁺, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn²⁺ compared to Mg²⁺. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn²⁺ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.