Abstract
In aerobically growing cells, in which reactive oxygen species are produced, the guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine as well as cytosine. This mispairing causes alterations in gene expression, and cells possess mechanisms to prevent such outcomes. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced by mutations in genes related to the prevention of abnormal protein synthesis under oxidative stress. A genome-wide search for the genes responsible, followed by DNA sequence determination, revealed that specific amino acid changes in guanylate kinase and in the β and β' subunits of RNA polymerase cause elevated levels of phenotypic suppression, specifically under aerobic conditions. The involvement of the DnaB, DnaN, and MsbA proteins, which are involved in DNA replication and in preserving the membrane structure, was also noted. Interactions of these proteins with each other and also with other molecules may be important for preventing errors in gene expression.
Highlights
Oxygen radicals, formed in aerobically growing cells, oxidize guanine to 8-oxo-7,8-dihydroguanine, which causes base mispairing
Partial Phenotypic Suppression of the lacZ Amber Mutation by 8-Oxo-Gua—Taddei et al [16] established the specific role of MutT protein in preventing transcriptional errors caused by 8-oxo-Gua by using the lacZ amber system developed by Cupples and Miller [20]. We have extended this system to allow for further identification of the genes required for accurate gene expression under oxidative stress
Errors in RNA synthesis can cause the accumulation of abnormal proteins, and a number of cellular functions are involved in preventing such outcomes
Summary
Oxygen radicals, formed in aerobically growing cells, oxidize guanine to 8-oxo-7,8-dihydroguanine, which causes base mispairing. We have applied a system related to partial phenotypic suppression using the lacZ amber mutation, which was previously used to establish the role of MutT in the prevention of transcriptional errors caused by oxidative damage [16] In this system, the action of an amber codon, UAG, was partially suppressed by the misincorporation of 8-oxo-Gua in place of uracil in the messenger RNA, which allows the formation of a small amount of active -galactosidase protein. Determining the sequences of the candidate genes, we would be able to identify the proteins responsible and further reveal specific amino acid residues necessary for executing these functions By performing these experiments, we found that certain base substitutions in the gmk and rpoC genes, encoding guanylate kinase and the Ј subunit of RNA polymerase, respectively, cause elevated levels of phenotypic suppression. We describe the results of these studies in detail and discuss the potential applications of this method, our findings, and the products developed during these studies
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