Purification and properties of the uracil DNA glycosylase from Bloom's syndrome.
Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.
Seal
G
G
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA.
Tallarida
R J
RJ
Sirover
M A
MA
D044383:Black People / Q000235:genetics; D001816:Bloom Syndrome / Q000201:enzymology* / Q000208:ethnology; D045647:DNA Glycosylases; D004795:Enzyme Stability; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes; D007585:Jews / Q000235:genetics*; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D011119:Polynucleotides / Q000378:metabolism; D013941:Thymine / Q000378:metabolism; D014481:United States / Q000208:ethnology; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D001741:Black or African American
D007527:Isoenzymes; D011119:Polynucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D013941:Thymine