Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poor folate status may be important in the aetiology of several epithelial cell malignancies including cancer of the uterine cervix. Folic acid is essential in the synthesis of purine nucleotides and the pyrimidine nucleoside thymidine and it is probable that imbalances in these DNA precursors negatively effect DNA stability and may ultimately lead to malignant transformation. The development of a modified 'comet assay' using the bacterial DNA repair enzyme uracil DNA glycosylase, to detect misincorporated uracil in human DNA is reported here. The effect of perturbing folic acid and deoxyuridine levels on uracil misincorporation in normal human lymphocytes and cultured human tumour cells was investigated using this assay. HeLa cells and peripheral human lymphocytes incubated as agarose-embedded nucleoids, with 1 unit of uracil DNA glycosylase per microg of DNA, contained low levels of uracil in their DNA. Both HeLa cells and stimulated human lymphocytes cultured in folate-deficient medium were growth arrested. Incubating human lymphocytes in folate-deficient medium significantly increased the level of uracil detected compared with control cells. HeLa cells showed an increase in non-specific DNA damage (strand breaks). Deoxyuridine (100 microM) significantly increased the level of uracil detected in the DNA of both folate-deficient and control HeLa cells. It appears that this modified comet assay specifically detects misincorporated uracil in single human cells. It should, therefore, prove valuable in determining the role of folic acid status in DNA instability and cancer.
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PMID:Uracil misincorporation in human DNA detected using single cell gel electrophoresis. 932 65

Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis). Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml-2 microgram/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil. Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1-10 ng/ml). Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results help to determine the mechanism(s) through which folic acid maintains DNA stability.
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PMID:DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro. 980 58

Changes in the folate and vitamin B12 status in the body influence the extent of uracil misincorporation (UrMis) into DNA, which is one of the biomarkers of genomic stability and, thus, portends a risk of cancer. In our study, the level of UrMis into DNA was evaluated by the comet assay (using the specific DNA repair enzyme, uracil DNA glycosylase) in leukocytes from blood donated by healthy young women with positive folate balance achieved by 4 weeks of folic acid supplementation (400 microg/day). The nutritional status was evaluated on the basis of nine food diaries recorded by the subjects during two winter months. The data were computerized, and the intake of nutrients and micronutrients was estimated using the DIETA 2 program (Food and Nutrition Institute, Warsaw, Poland) linked to recently updated Polish food tables. The plasma folate and vitamin B12 concentration, as well as methylenetetrahydrofolate reductase (MTHFR) polymorphisms, were evaluated to determine their influence on the level of UrMis into DNA. The mean value of B12 intake for all subjects reached 100% of the Polish recommended dietary allowances (RDA), whereas the mean value of folate intake, before folate supplementation, was 50%, suggesting moderate deficiency. Folic acid supplementation brought the folate intake way above the RDA, and plasma folate concentration in each individual was above the deficient range (mean value 14.67 ng/ml). The UrMis did not correlate with the plasma folate concentration, but the level of UrMis was significantly lower in subjects with plasma vitamin B12 concentration above 400 pg/ml (P=.02) only after folic acid supplementation. The concentration of folate in plasma correlated (P<or=.05) with the wild-type MTHFR homozygote 1298 AA but not with the MTHFR 677 genotype. When subjects were grouped according to genotype, the mean concentration of folate in plasma was significantly lower in subjects with the MTHFR 677 (CT+TT) polymorphism, which was accompanied by a lower UrMis, compared to individuals with the CC genotype. The significantly higher concentrations of folate in serum, accompanied by increased UrMis, were seen in subjects with the combined MTHFR 1298 (AC+CC) genotype, as compared to the 1298 AA wild type. Our results suggest that more than 400 pg/ml of vitamin B12 in plasma in subjects with a positive folate balance is critical for genomic stability and indicate that the amount of UrMis into DNA is related to the MTHFR genotype.
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PMID:Uracil misincorporation into DNA of leukocytes of young women with positive folate balance depends on plasma vitamin B12 concentrations and methylenetetrahydrofolate reductase polymorphisms. A pilot study. 1604 29