Elimination and utilization of oxidized guanine nucleotides in the synthesis of RNA and its precursors

Abstract

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

FIGURE 1.

SDS-PAGE of purified preparations of E. coli enzymes. Purified proteins (1 μg each) were subjected to 5–20% SDS-PAGE. Lane 1, molecular mass markers; lane 2, N-terminally His-tagged MutT; lane 3, GST-guanylate kinase fusion protein; lane 4, GST-nucleoside-diphosphate kinase fusion protein; lane 5, GST-adenylate kinase fusion protein.

FIGURE 2.

Actions of guanylate kinase on nucleoside monophosphates containing normal and oxidized guanine bases. A, the action of GMK on GMP. The reaction mixture (20 μl) contained 0.2 mm GMP, 0.2 mm ATP, 50 mm Hepes (pH 7.4), 4 mm MgCl₂, 40 mm (NH₄)₂SO₄, and 20 ng of a purified preparation of GMK. The reaction was carried out at 37 °C for 8 min, and then samples were applied to HPLC using a TSKgel DEAE-2 SW column (Tosoh, Tokyo, Japan). B, the action of GMK on 8-oxo-GMP. The reaction was performed as described above except that 8-oxo-GMP was treated with 1 μg of the GMK protein for 30 min. C, the time courses of reactions for GMP (●) and 8-oxo-GMP (○) with GMK. D, the time courses of reactions for dGMP (●) and 8-oxo-dGMP (○) with GMK. For C and D, the reactions were carried out with 20 ng of GMK preparation and 0.2 mm substrate nucleotides in 20 μl of a reaction mixture containing 50 mm Hepes (pH 7.4), 0.2 mm ATP, 4 mm MgCl₂, and 40 mm (NH₄)₂SO₄.

FIGURE 3.

Actions of nucleoside diphosphate kinase on nucleoside diphosphates containing normal and oxidized guanine bases. A, the actions of NDK on GDP. The reaction mixture (10 μl) contained 0.5 mm GDP, 0.5 mm ATP, 50 mm Hepes (pH 7.4), 4 mm MgCl₂, 40 mm (NH₄)₂SO₄, and 100 ng of a purified preparation of NDK. The reaction was carried out at 37 °C for 4 min, and the reaction products were analyzed by HPLC. B, the actions of NDK on 8-oxo-GDP. The reaction was performed as described above. C, the time courses of reactions for GDP (●) and 8-oxo-GDP (○) with NDK. D, the time course of reactions for dGDP (●) and 8-oxo-dGDP (○) with NDK. For C and D, the reactions were carried out with 100 ng of NDK preparation and 0.5 mm substrate nucleotides in 10 μl of a reaction mixture containing 50 mm Hepes (pH 7.4), 0.5 mm ATP, 4 mm MgCl₂, and 40 mm (NH₄)₂SO₄.

FIGURE 4.

Time courses of reactions for GDP and 8-oxo-GDP with adenylate kinase. The reaction was carried out with 800 ng of ADK preparation and 0.2 mm GDP (●) or 8-oxo-GDP (○) in a reaction mixture (10 μl) containing 50 mm Hepes (pH 7.4), 0.2 mm ADP, 4 mm MgCl₂, and 40 mm (NH₄)₂SO₄.

FIGURE 5.

Selective degradation of 8-oxoGTP by MutT in the presence of a large amount of ATP, GTP, CTP, and UTP. The reaction mixture (25 μl) contained 1 μm 8-oxo-[¹⁴C]GTP, 0.9 mm GTP, 3 mm ATP, 0.52 mm CTP, 0.89 mm UTP, 1 ng of MutT, 20 mm Tris-HCl (pH 8), 8 mm MgCl₂, 40 mm NaCl, 5 mm DTT, 80 μg/ml BSA, and 2% (v/v) glycerol. The reaction was performed at 30 °C and terminated at 0 or 30 min by adding SDS (final concentration, 0.1%). The samples were resolved by HPLC with a Mono Q column. Nucleotides were separated with a linear gradient (5–100%) of 1 m triethylammonium hydrogen carbonate (pH 7.0) at a flow rate of 1 ml/min using HPLC.

FIGURE 6.

Metabolism of guanine ribonucleotides leading to the synthesis of RNA under normal and oxidative conditions. RNP denotes RNA polymerase. O^• indicates reactive oxygen species, and the closed dots to the upper left of chemicals represent 8-oxoguanine-containing molecules.