Search for proteins required for accurate gene expression under oxidative stress: roles of guanylate kinase and RNA polymerase.

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 ß and ß' 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.

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

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.

Human MTH3 (NUDT18) protein hydrolyzes oxidized forms of guanosine and deoxyguanosine diphosphates: comparison with MTH1 and MTH2.

Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.

Cleavage of oxidized guanine nucleotide and ADP sugar by human NUDT5 protein.

MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.

Molecular actions of Escherichia coli MutT for control of spontaneous mutagenesis.

MutT protein of Escherichia coli hydrolyzes oxidized guanine nucleotides, 8-oxo-dGTP and 8-oxoGTP, to the corresponding monophosphates, thereby preventing misincorporation of 8-oxoguanine into DNA and RNA, respectively. Although the biological significance of the MutT has been established, how MutT protein actually works in vivo remains to be elucidated. The current study shows the molecular behavior of the MutT protein in vivo and in vitro with special reference to control of spontaneous mutagenesis. A single E. coli cell carries about 70-75 molecules of the MutT protein and that this number does not change even when the cells were cultured in anaerobic and hyper-oxidative conditions. Conditional gene silencing analyses revealed that about a half number of MutT molecules are needed for keeping the spontaneous mutation frequency at the normal level. The MutT functions are not needed under anaerobic condition, yet the level of the MutT protein in cell is kept constant, probably for preparing for sudden changes of oxygen pressure. There is a possibility that MutT functions in close association with other proteins, and evidence is presented that MutT protein can interact with some proteins in vivo.

Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress.

Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage.

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with V(max)/K(m) values of 1.3 x 10(-3) and 1.7 x 10(-3), respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1-0.5 x 10(-3)). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.

Multiple enzyme activities of Escherichia coli MutT protein for sanitization of DNA and RNA precursor pools.

8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acids as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. MutT protein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to corresponding nucleoside monophosphates, thereby preventing misincorporation of 8-oxoGua into DNA and RNA, which would cause mutation and phenotypic suppression, respectively. Here, we report that the MutT protein has additional activities for cleaning up the nucleotide pools to ensure accurate DNA replication and transcription. It hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP with a K(m) of 0.058 microM, a value considerably lower than that for its normal counterpart, dGDP (170 microM). Furthermore, the MutT possesses an activity to degrade 8-oxo-GDP to the related nucleoside monophosphate, with a K(m) value 8000 times lower than that for GDP. These multiple enzyme activities of the MutT protein would facilitate the high fidelity of DNA and RNA syntheses.

Killing and mutagenic actions of dacarbazine, a chemotherapeutic alkylating agent, on human and mouse cells: effects of Mgmt and Mlh1 mutations.

Among various types of drugs designed for use in cancer chemotherapy, some have the potential for alkylation. After metabolic activation, these chemicals attack DNA and alkylate their bases, thereby preventing multiplication of rapidly growing tumor cells. Some of alkylated bases cause mutations, leading to untoward induction of tumors. To search for the rationale to separate lethal and mutagenic effects of alkylation drugs, we investigated actions of dacarbazine, a monofunctional triazene, on mouse and human cell lines defective in the Mgmt and/or the Mlh1 gene, the former encoding a DNA repair methyltransferase and the latter a protein involved in mismatch repair and induction of apoptosis. Mgmt-deficient cells are hypersensitive to the killing action of dacarbazine. On the other hand, cells defective in both Mgmt and Mlh1 genes are as resistant to the drug as are wild-type cells, in terms of survival, but do have many mutations after dacarbazine treatment. Thus, the killing and mutagenic actions of dacabazine can be dissociated by manipulating actions of these gene products.

Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis.

To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt(-/-) cells are hypersensitive to the killing effect of N-methyl-N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt(-/-)Mlh1(-/-) cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt(-/-)Mlh1(-/-) cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt(-/-)Mlh1(-/-) cells became 10-fold higher after being treated with MNU. Mgmt(-/-)Mlh1(+/-) cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt(-/-)Mlh1(+/-) cells were as resistant to MNU as were Mgmt(-/-)Mlh1(-/-) cells. While caspase-3 was induced in Mgmt(-/-)Mlh1(+/+) cells after treatment with MNU, no induction occurred in Mgmt(-/-)Mlh1(+/-) cells or in Mgmt(-/-)Mlh1(-/-) cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues.