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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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Register-Shifted Structures in Base-Flipped Uracil-Damaged DNA
We report the occurrence of register shifted structures in simulations of uracil-containing dsDNA. These occur when the 3' base vicinal to uracil is thymine in U:A base paired DNA. Upon base flipping of uracil, this 3' thymine hydrogen bonds with the adenine across the uracil instead of its complementary base. The register shifted structure is persistent, and sterically blocks re-entry of uracil into the helix stack. Register shifting might be important for DNA repair, since the longer exposure of the lesion in register shifted structures could facilitate enzymatic recognition and repair.
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- Award ID(s):
- 1919096
- PAR ID:
- 10548677
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- Journal of the American Chemical Society
- Volume:
- 145
- Issue:
- 30
- ISSN:
- 0002-7863
- Page Range / eLocation ID:
- 16350 to 16354
- 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/10.1021/jacs.3c05890
