Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.
We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.
UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
I
I
Eftedal
P H
PH
Guddal
G
G
Slupphaug
G
G
Volden
H E
HE
Krokan
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D014498:Uracil; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase