Cargando…
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 t...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8861164/ https://www.ncbi.nlm.nih.gov/pubmed/35085553 http://dx.doi.org/10.1016/j.jbc.2022.101638 |
_version_ | 1784654828819972096 |
---|---|
author | Hsu, Chia Wei Sowers, Mark L. Baljinnyam, Tuvshintugs Herring, Jason L. Hackfeld, Linda C. Tang, Hui Zhang, Kangling Sowers, Lawrence C. |
author_facet | Hsu, Chia Wei Sowers, Mark L. Baljinnyam, Tuvshintugs Herring, Jason L. Hackfeld, Linda C. Tang, Hui Zhang, Kangling Sowers, Lawrence C. |
author_sort | Hsu, Chia Wei |
collection | PubMed |
description | 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 oligonucleotides 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 formation, persistence, and repair of a biologically important class of deaminated cytosine adducts. |
format | Online Article Text |
id | pubmed-8861164 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-88611642022-02-27 Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase Hsu, Chia Wei Sowers, Mark L. Baljinnyam, Tuvshintugs Herring, Jason L. Hackfeld, Linda C. Tang, Hui Zhang, Kangling Sowers, Lawrence C. J Biol Chem Research Article 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 oligonucleotides 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 formation, persistence, and repair of a biologically important class of deaminated cytosine adducts. American Society for Biochemistry and Molecular Biology 2022-01-25 /pmc/articles/PMC8861164/ /pubmed/35085553 http://dx.doi.org/10.1016/j.jbc.2022.101638 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Article Hsu, Chia Wei Sowers, Mark L. Baljinnyam, Tuvshintugs Herring, Jason L. Hackfeld, Linda C. Tang, Hui Zhang, Kangling Sowers, Lawrence C. Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title | Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title_full | Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title_fullStr | Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title_full_unstemmed | Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title_short | Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase |
title_sort | measurement of deaminated cytosine adducts in dna using a novel hybrid thymine dna glycosylase |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8861164/ https://www.ncbi.nlm.nih.gov/pubmed/35085553 http://dx.doi.org/10.1016/j.jbc.2022.101638 |
work_keys_str_mv | AT hsuchiawei measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT sowersmarkl measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT baljinnyamtuvshintugs measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT herringjasonl measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT hackfeldlindac measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT tanghui measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT zhangkangling measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase AT sowerslawrencec measurementofdeaminatedcytosineadductsindnausinganovelhybridthyminednaglycosylase |