Purification and characterization of 5-hydroxymethyluracil-DNA glycosylase from calf thymus. Its possible role in the maintenance of methylated cytosine residues.
5-Hydroxymethyluracil (HmUra) residues formed by the oxidation of thymine are removed from DNA through the action of a DNA glycosylase activity. This activity was purified over 1870-fold from calf thymus and found to be distinct from uracil (Ura)-DNA glycosylase. The HmUra-DNA glycosylase has a molecular weight of 38,000, a pH optimum of 6.7-6.8 and an apparent Km of 0.73 +/- 0.04 microM. These values are similar to those reported for other mammalian DNA glycosylases. The enzyme removed HmUra residues from single- and double-stranded DNA with almost equal efficiency. HmUra-DNA glycosylase activity was not product inhibited by free HmUra. The DNA glycosylase activity was inhibited by Mg2+, but the purest enzyme fractions contained a Mg2+-dependent apurinic/apyrimidinic endonuclease activity. HmUra-DNA glycosylase and the recently described 5-hydroxymethylcytosine (HmCyt)-DNA glycosylase (Cannon, S. V., Cummings, A. C., and Teebor, G. W. (1988) Biochem. Biophys. Res. Commun. 151, 1173-1179) are unique among known DNA glycosylases in being present in mammalian cells and absent from bacteria. These DNA glycosylase activities were shown here to reside on different proteins. We suggest that the major function of HmUra-DNA glycosylase, together with HmCyt-DNA glycosylase, is the maintenance of methylated cytosine residues in the DNA of higher organisms.
Cannon-Carlson
S V
SV
Department of Pathology, New York University School of Medicine 10016.
Gokhale
H
H
Teebor
G W
GW
D000818:Animals; D002417:Cattle; D003596:Cytosine / Q000378:metabolism*; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000502:physiology; D051381:Rats; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D014498:Uracil / Q000031:analogs & derivatives*; D051981:Uracil-DNA Glycosidase
C008055:5-hydroxymethyluracil; D014498:Uracil; D010432:Pentoxyl; D003596:Cytosine; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase