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Increased expression of the human telomere reverse transcriptase (hTERT) in tumors promotes tumor cell survival and diminishes the survival of patients. Cytosine-to-thymine (C-to-T) transition mutations (C250T or C228T) in the hTERT promoter create binding sites for transcription factors, which enhance transcription. The G-rich strand of the hTERT promoter can form G-quadruplex structures, whereas the C-rich strand can form an i-motif in which multiple cytosine residues are protonated. We considered the possibility that i-motif formation might promote cytosine deamination to uracil and C-to-T mutations. We computationally probed the accessibility of cytosine residues in an i-motif to attack by water. We experimentally examined regions of the C-rich strand to form i-motifs using pH-dependent UV and CD spectra. We then incubated the C-rich strand with and without the G-rich complementary strand DNA under various conditions, followed by deep sequencing. Surprisingly, deamination rates did not vary substantially across the 46 cytosines examined, and the two mutation hotspots were not deamination hotspots. The appearance of mutational hotspots in tumors is more likely the result of the selection of sequences with increased promoter binding affinity and hTERT expression.
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Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase
The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oli- gonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the for- mation, persistence, and repair of a biologically important class of deaminated cytosine adducts.
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- Award ID(s):
- 2129617
- PAR ID:
- 10482358
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 298
- Issue:
- 3
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 101638
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.jbc.2022.101638
