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

Abstract

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.

Keywords: RNA extension; enzyme kinetics; human polymerase η; manganese; translesion synthesis.

Figure 1

Manganese activates the RNA synthetic activity of hPolη. (A) Primer extension reactions were performed applying two different concentrations of the indicated divalent cations. The structure of the primer/template used in the experiments is shown on the top. The black line depicts a DNA strand, and the red line is an RNA strand. The asterisk indicates a fluorescent label. The first templating nucleotide is shown. Reactions contained 30 nM Polη, 20 nM primer/template, and either 50 μM of dNTPs (left panel) or 1 mM of rNTPs (right panel). The positions of the primer and its one nucleotide extension are indicated. (B) Mg²⁺ and Mn²⁺ concentration-dependent RNA extension. Reactions were performed for 10 min with 20 nM hPolη in the presence of 20 nM RNA/DNA and 1 mM rNTPs. The concentrations of Mn²⁺ and Mg²⁺ are indicated below each lane.

Figure 2

The binding affinity of hPolη to DNA and RNA primers in the presence of Mg²⁺ or Mn²⁺. (A) DNA/DNA and RNA/DNA primer/templates (20 nM) were incubated with 0 to 350 nM hPolη as indicated, in the presence of 4 mM Mg²⁺ or Mn²⁺. Complexes were resolved on a 4% non-denaturing polyacrylamide gel. (B) Quantitation of binding affinities. 10 nM of DNA/DNA or RNA/DNA was incubated with hPolη (from 22 to 175 nM in 11 increments). The standard deviation (SD) for each substrate is indicated.

Figure 3

Manganese enhances the velocity of the polymerization reaction. Primer extension reactions were performed with 5 nM hPolη, 20 nM RNA/DNA, 500 μM of individual rNTPs, and 4 mM Mg²⁺ or Mn²⁺ for the indicated times. Labels are the same as in Figure 1.

Figure 4

Manganese enhances hPolη’s affinity to ribonucleotides. Reactions were performed using 1 nM enzyme, 20 nM RNA/DNA, 4 mM Mg²⁺ or Mn²⁺, and various concentrations of individual rNTPs as indicated. Reaction time was 45 min. Labels are the same as in Figure 1.

Figure 5

Fidelity of hPolη in the presence of magnesium or manganese. (A) To examine the fidelity of DNA synthesis, reactions were run with 3 nM hPolη, 20 nM DNA/DNA, 100 μM individual dNTP, and 4 mM Mg²⁺ or Mn²⁺, as indicated, for 1 min. The first templating nucleotide is shown above each panel and the correct incoming nucleotides are framed below the pictures. (B) RNA synthesis reactions were performed as in (A) except using 20 nM RNA/DNA and 2000 μM rNTP for 15 min. (C) Time course reactions were performed with 1 nM hPolη, 20 nM RNA/DNA, 4000 μM of individual rNTPs, and 4 mM Mn for the indicated times.

Figure 6

DNA damage bypass by hPolη during RNA synthesis using magnesium or manganese. (A–D) Bypass of 8-oxoG: (A) To check the velocity of bypass, 2 nM hPolη was incubated with 8 nM RNA/DNA and 500 μM of rCTP for the indicated times. The asterisk indicates a fluorescent label. (B) The affinity of hPolη to rCTP during bypass was tested using 1 nM enzyme and 8 nM RNA/DNA for 45 min with various concentrations of rCTP as indicated below each lane. (C) The fidelity of oxo-G bypass was examined using 8 nM RNA/DNA, 500 μM rNTP, and either 3 nM hPolη for 45 min (left panel) or 2 nM hPolη for 30 min (right panel). (D) A time course of misincorporation in the presence of manganese was performed with 0.8 nM hPolη, 8 nM RNA/DNA, and 4000 μM of individual rNTPs for the times indicated above each lane. In (C,D), the percentages of extended primers are shown below each lane. The correct incoming rCTP is boxed. (E–H) Bypass of TT dimer. (E) Bypass was assayed with 4 nM hPolη, 16 nM RNA/DNA, and 500 μM rATP for the indicated times. (F) Reactions contained 1 nM enzyme, 16 nM RNA/DNA, and various concentrations of rATP, as indicated below each lane. Incubation time was 45 min (G) Reactions were performed using 16 nM RNA/DNA, 500 μM individual rNTPs, and 3 nM hPolη for 15 min. (H) A time course of misincorporation in the presence of manganese was run with 1 nM hPolη, 16 nM RNA/DNA, and 500 μM of individual rNTPs for the times indicated above each lane. In (E–H) labels are the same as on (A–D).