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. 2000 Dec 19;97(26):14183-7.
doi: 10.1073/pnas.97.26.14183.

An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts

V Rusmintratip  1 , L C Sowers
Affiliations

An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts

V Rusmintratip et al. Proc Natl Acad Sci U S A. .

Abstract

The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10(7). The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.

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Figures

Figure 1
Figure 1
Sequence of the oligonucleotides used in this assay, where X = U, T, HmU, C, 5mC, or HmC and where P = A or G.
Figure 2
Figure 2
Polyacrylamide gel of oligonucleotides following incubation with HeLa cell nuclear extract. The sequence of the duplex is indicated in Fig. 1. The identity of the base pair at the cleavage site is indicated in the figure. Lanes marked HmU-control and HmU ss refer to the HmU-containing single strand not treated with extract, and the HmU-containing single strand treated with extract.
Figure 3
Figure 3
Cleavage of duplex containing HmU:G base pair at target site treated with HeLa cell nuclear extract for the periods of time indicated in the figure.
Figure 4
Figure 4
Plot of the amount of duplex containing an HmU:G or HmU:A base pair cleaved per microgram of protein as a function of time.
Scheme 1
Scheme 1
Pathways by which HmU:G and HmU:A base pairs may be formed in DNA.
Figure 5
Figure 5
Polyacrylamide gel of oligonucleotides following incubation with human fibroblast whole cell extract. The sequence of the duplex is indicated in Fig. 1. The identity of the base pair at the cleavage site is indicated in the figure.
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