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.
Highlights
Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides
Action of Guanylate Kinase on Oxidized GMP—The 8-oxoGua-containing ribonucleoside monophosphate 8-oxo-GMP can be formed by the direct oxidation of GMP by Reactive oxygen species (ROS) and by the cleavage of 8-oxo-GDP and 8-oxo-GTP by the MutT protein
The E. coli guanylate kinase was produced in cells harboring multicopy plasmids expressing the gmk gene, and a nearly homogeneous enzyme preparation was obtained
Summary
Bacterial Strains—E. coli strain CC101 and its mutTϪ deficient derivative, CC101T, were used in these studies [13, 15]. Fractions containing 8-oxo-GTP were combined and applied again to the Mono Q column to remove unoxidized nucleotides. The cells were treated with 0.5 mM isopropyl -D-thiogalactoside at 37 °C for 2 h to induce the expression of the GST-NDK protein, which was purified as described above. Purification of ADK—Cloning of the E. coli ADK gene (adk/ plsA/dnaW) was performed by PCR to amplify DNA fragments from E. coli strain CC101 using a primer set for ADK (forward primer, 5Ј-GGGGATCCATGCGTATCATTCTGCTT-3Ј; reverse primer, 5Ј-GGCTCGAGTTAGCCGAGGATTTTTTCC-3Ј) as described above. A total of 5 ml of overnight culture of DH5␣ cells harboring pGEX-KG-ADK was mixed with 100 ml of LB broth and grown to an A540 of 0.2 at 37 °C. The cells were treated with 0.5 mM isopropyl -D-thiogalactoside at 37 °C for 2 h to induce the expression of the GST-ADK protein, which was purified as described above.
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