The precise role of vitamin C in the prevention of DNA mutations is controversial. Although ascorbic acid has strong antioxidant properties, it also has pro-oxidant effects in the presence of free transition metals. Vitamin C was recently reported to induce the decomposition of lipid hydroperoxides independent of metal interactions, suggesting that it may cause DNA damage. To directly address the role of vitamin C in maintaining genomic integrity we developed a genetic system for quantifying guanine base mutations induced in human cells under oxidative stress. The assay utilized a plasmid construct encoding the cDNA for chloramphenicol acetyl transferase modified to contain an amber stop codon, which was restored to wild type by G to T transversion induced by oxidative stress. The mutation frequency was determined from the number of plasmids containing the wild type chloramphenicol acetyl transferase gene rescued from oxidatively stressed cells. Cells were loaded with vitamin C by exposing them to dehydroascorbic acid, thereby avoiding transition metal-related pro-oxidant effects of ascorbic acid. We found that vitamin C loading resulted in substantially decreased mutations induced by H 2 O 2 . Depletion of glutathione led to cytotoxicity and an increase in H 2 O 2 -induced mutation frequency; however, mutation frequency was prominently decreased in depleted cells preloaded with vitamin C. The mutation results correlated with a decrease in total 8-oxo-guanine measured in genomic DNA of cells loaded with vitamin C and oxidatively stressed. These findings directly support the concept that high intracellular concentrations of vitamin C can prevent oxidation-induced mutations in human cells.DNA damage caused by reactive oxygen species such as H 2 O 2 , O 2 . , and ⅐ OH radicals has been implicated in mutagenesis, oncogenesis, and aging (1). Oxidative lesions in DNA include base modifications, sugar damage, strand breaks, and abasic sites. In vitro studies suggest that the hydroxyl radical is highly reactive toward DNA (2). One of the most common oxidized adducts in human cells and tissues is 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (8-oxo-dG) 1 (3). This adduct results from exposure to oxidizing agents as well as from ␥ irradiation of DNA (4), and quantitation of 8-oxo-dG has been used as a marker of DNA damage (5). 8-oxo-dG "mis-pairs" with adenine during replication (6), resulting in G to T transversions in 50% of the replicated DNA (7).The role of vitamin C in protecting against oxidatively induced DNA mutations is controversial. Although numerous studies demonstrate the antioxidant effects of vitamin C (8, 9), in vitro studies are often confounded by the pro-oxidant effects of ascorbic acid in the presence of free transition metals (10). We circumvented this problem using dehydroascorbic acid to load cells with vitamin C. Vitamin C is transported into most cells in the oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (11, 12) and as ascorbic acid in specialized cells by sodium-dependen...
The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U⅐G mismatches (Gallinari, P., and Jiricny, J. (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N 4 -ethenocytosine (⑀C) from ⑀C⅐G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508 -8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U⅐G and T⅐G mismatches, respectively, we conclude that Mug does not repair U⅐G or T⅐G mismatches in vivo. A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts from mug ؉ ung cells show very little ability to remove uracil from DNA, but can excise ⑀C. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.Cytosine is the most unstable of the four bases in DNA and deaminates hydrolytically to create U⅐G mismatches. If unrepaired, uracil can pair with an adenine during replication causing a C to T mutation. For this reason, cells contain uracil-DNA glycosylase (Ung), an enzyme that removes the uracil and initiates its replacement with cytosine. The importance of Ung in mutation avoidance is evidenced by the observation that ung strains of Escherichia coli (1) and yeast (2) accumulate C to T mutations.Cytosines methylated at position 5 similarly deaminate to create T⅐G mismatches, which are not subject to repair by Ung. In E. coli, a specialized mismatch correction process called very short patch repair corrects these mispairs to C⅐G (3). The key enzyme in this repair pathway is a sequence-specific, mismatch-specific endonuclease, Vsr, which hydrolyzes the phosphodiester linkage preceding the mismatched T (4). No eukaryotic sequence homologs of this enzyme have been reported; instead a DNA glycosylase is thought to serve the same function (5). This enzyme excises thymines from T⅐G mismatches (6) and prefers mismatches that are followed by a G⅐C pairs (7,8). This enzyme, thymine-DNA glycosylase (TDG), 1 could prevent mutations when 5-methylcytosines within CG dinucleotides deaminate to thymine.The cDNA for TDG was cloned and its sequence was determined (9). Remarkably, a sequence homolog of this protein was found in E. coli and Serratia ma...
Vitamin C is transported as ascorbic acid (AA) through the sodium-ascorbate cotransporters (SVCT1 and -2) and as dehydroascorbic acid (DHA) through the facilitative glucose transporters. All cells have glucose transporters and take up DHA that is trapped intracellularly by reduction and accumulated as AA. SVCT2 is widely expressed in cells and tissues at the mRNA level; however, only specialized cells directly transport AA. We undertook a molecular analysis of SVCT2 expression and discovered a transcript encoding a short form of human SVCT2 (hSVCT2-short) in which 345 bp is deleted without a frame shift. The deletion involves domains 5 and 6 and part of domain 4. cDNA encoding this isoform was isolated and expressed in 293T cells, where the protein was detected on the plasma membrane. Transport studies, however, revealed that hSVCT2-short gave rise to a nonfunctional transporter protein. hSVCT2-short arises by alternative splicing and encodes a protein that strongly inhibited the function of SVCT2 and, to a lesser extent, SVCT1 in a dominant-negative manner, probably by protein-protein interaction. The expression of hSVCT2-short varies among cells. PCR analysis of cDNA isolated from melanocytes capable of transporting AA revealed a predominance of the full-length isoform, while HL-60 cells, which express SVCT2 at the mRNA level and were incapable of transporting AA, showed a predominance of the short isoform. These findings suggest a mechanism of AA uptake regulation whereby an alternative SVCT2 gene product inhibits transport through the two known AA transporters.Vitamin C is essential for human health. Most mammals produce vitamin C in the liver; however, humans and other primates are unable to synthesize ascorbic acid (AA) and must obtain it from the diet (9, 13). Vitamin C is transported into cells in the oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (GLUTs) (19,24) and as AA in specialized cells by sodium-dependent AA transporters (23). Two isoforms of the sodium-dependent vitamin C transporters (SVCTs) have been molecularly characterized in rats and humans (3,10,18,23,28,29). Kyte-Doolittle hydropathy analysis (7) of the human SVCT2 (hSVCT2) amino acid sequence predicts a topographical model of a transporter with 12 transmembrane domains with both the N and C termini intracellular. The N-terminal (102-amino-acid) and the C-terminal (81-amino-acid) tails in the cytoplasm are long and hydrophilic. The extracellular loop between transmembrane domains 3 and 4 contains two potential sites for N-glycosylation . The hSVCT1 transporter is highly homologous to hSVCT2 with the same predicted membrane topology. An obvious difference between hSVCT1 and hSVCT2 is the additional sequences of 12 and 44 amino acids present in the N terminus of hSVCT2 at positions 2 and 38, respectively (10). The two isoforms of hSVCT differ in tissue distribution, as determined by Northern blot analysis, with hSVCT2 widely expressed at the mRNA level compared to hSVCT1. For hSVCT2, a 7.5-kb transcript wa...
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