Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase

Abstract

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.

Somatic Hypermutation and Class Switch Recombination in Msh6−/−Ung−/− Double-Knockout Mice

“…This scarcity of A,T mutations in mice deficient for Msh6 alone disagrees with the conclusion that Ung is responsible for a proportion of up to 52% of mutations from A and T. However, this finding hints at a collaboration between Ung and MMR (26). Ung is more active on ssDNA than dsDNA (36,37). Presumably, in normal SHM, Ung acts efficiently on ssDNA created by Msh2/Msh6 induced excision of either the U or G containing strand.…”

Inefficient excision of uracil from loop regions of DNA oligomers byE.coliuracil DNA glycosylase

“…Slightly better release of uracil from a G:U pair is also relevent from physiological considerations where cytosine deamination will result in a G:U pair. Thus a more recent report (7) showing somewhat less efficient excision of uracil from a G:U pair (compared to an A:U pair) is still not understood. During these studies we found that excision of uracil from tetraloop regions of hairpin structures (U-loop, rp-ung, Table 2) and a penta-loop region (U-loopUS, Figure 6) is very inefficient.…”

“…However, the latter is kept to a minimum by the presence of dUTPase (dut) which maintains low levels of dUTP in the cell (1)(2)(3). UNG uses single-stranded substrates more efficiently than double-stranded substrates (4)(5)(6)(7). However, it was also reported (8) that uracil residues from some regions of single-stranded M13 DNA were removed inefficiently.…”

DNA glycosylases in the base excision repair of DNA

“…The rate differs as much as 20-fold between the ' best ' (A\T\UAA) and ' worst ' (G\CUG\C\T) sequences. Usually, but not always [34], the rate of removal is greater from U\G mispairs than from U :A pairs [35][36][37]. A low removal rate tends to be correlated with the occurrence of ' hot spots ' for mutation [36], similar to the correlation between slow spots for the repair of cyclobutyl dimers and hot spots for mutations [38,39].…”