Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase.
We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.
Slupphaug
G
G
UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Eftedal
I
I
Kavli
B
B
Bharati
S
S
Helle
N M
NM
Haug
T
T
Levine
D W
DW
Krokan
H E
HE
D001483:Base Sequence; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase