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

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. Oxidative DNA damage has been implicated in cancer and aging (1-3). The oxidation of the thymine methyl group of a T:A base pair can generate 5-hydroxymethyluracil (HmU; refs. 4 and 5). The biological implications of this DNA damage are currently unclear; however, the laboratories of Ames (6) and Teebor (7) have identified a specific glycosylase in higher eukaryotes that removes HmU from an HmU:A base pair in DNA. The need for DNA repair activities is generally discussed within the context of removing lesions that miscode or block the progression of a DNA or RNA polymerase. However, HmU is not miscoding (8), it does not perturb DNA structure (9, 10), and it does not impede polymerases (11,12). Indeed, in some bacteriophage, HmU completely replaces T (13).In the absence of an obvious need for the removal of HmU, the existence of the HmU-DNA glycosylase activity led to the proposal that the role of this activity was to remove HmU that could arise in DNA by a second pathway-the oxidation and deamination of 5-methylcytosine (5mC; refs. 7, 14, and 15). In support of this hypothesis, it was demonstrated that the HmU-DNA glycosylase is found only in higher eukaryotes that have 5mC in the genome (16). The methylated CpG dinucleotide is a reactive center for several carcinogens (17-19), and transition mutations at this site are frequently observed in human tumors (20 -22). Recently, the apparent involvement of altered cytosine methylation patterns in the development of human cancer has renewed interest in understanding chemical mechanisms that perturb cytosine methylation patterns (23)(24)(25)(26)(27).In the studies that initially identified HmU-DNA glycosylase activity, the substrate was the DNA of HmU-containing bacteriophage (6, 7). In these bacteriophage, the HmU replaces T and therefore pairs with A. Because HmU pairs with only A during polymerase-directed DNA synthesis, the preparation ...

DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria

“…It has been proposed that 5hmC is an intermediate metabolite in active demethylation of the genome by repair enzymes (30), in passive demethylation as a result of lack of recognition by enzymes involved in maintenance methylation (31), or that it alters local chromatin structure because 5hmC is not recognized by 5-methylcytosine-binding proteins (7). The role of 5hmC in the mitochondrial genome likely involves one or more of these processes.…”

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

“…It is possible there are still other glycosylases in human cells that have the ability to remove HmU from the DNA. In these and other studies (42,43), characterization of repair activities against damaged bases have made use of purified glycosylases or cell nuclear extracts in conjunction with base excision assays. However, the method we present here using stable-isotope tracers is able to measure repair rates in living cells against an important oxidative DNA damage product while simultaneously monitoring DNA replication, leading to a more global picture of base excision repair rates in living cells.…”

5-Hydroxymethylcytosine Profiling as an Indicator of Cellular State

“…Alternatively, it has been suggested that demethylation occurs through deamination of 5hmC to 5-hydroxymethyluracil with subsequent base-excision repair, resulting in a nonmodified CpG dinucleotide [61][62][63]. Once more, the evidence supporting such a pathway has also been called into question.…”