Definitive characterization of human thymine glycol N-glycosylase activity.
An N-glycosylase activity that released cis-[3H]-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-[3H]dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III. This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH). The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-[14C]TG and [14C]HMH, which had been independently synthesized, as reference compounds. This technique facilitated construction of a v/[E]t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity. The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA.
Higgins
S A
SA
Frenkel
K
K
Cummings
A
A
Teebor
G W
GW
D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification; D011067:Poly dA-dT / Q000138:chemical synthesis; D013379:Substrate Specificity; D014316:Tritium
D002250:Carbon Radioisotopes; D029968:Escherichia coli Proteins; D014316:Tritium; D011067:Poly dA-dT; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases