URACIL MISINCORPORATION IN HUMAN DNA DETECTED USING SINGLE-CELL GEL-ELECTROPHORESIS

Poor folate status may be important in the aetiology of several epithe lial cell malignancies including cancer of the uterine cervix. Folic a cid is essential in the synthesis of purine nucleotides and the pyrimi dine 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 ass ay' using the bacterial DNA repair enzyme uracil DNA glycosylase, to d etect misincorporated uracil in human DNA is reported here, The effect of perturbing folic acid and deoxyuridine levels on uracil misincorpo ration in normal human lymphocytes and cultured human tumour cells was investigated using this assay, HeLa cells and peripheral human lympho cytes incubated as agarose-embedded nucleoids, with 1 unit of uracil D NA glycosylase per mu g of DNA, contained low levels of uracil in thei r DNA, Both HeLa cells and stimulated human lymphocytes cultured in fo late-deficient medium were growth arrested. Incubating human lymphocyt es in folate-deficient medium significantly increased the level of ura cil detected compared with control cells, HeLa cells showed an increas e in non-specific DNA damage (strand breaks), Deoxyuridine (100 mu M) significantly increased the level of uracil detected in the DNA of bot h folate-deficient and control HeLa cells, It appears that this modifi ed comet assay specifically detects misincorporated uracil in single h uman cells, It should, therefore, prove valuable in determining the ro le of folic acid status in DNA instability and cancer.