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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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Divergence in dimerization and activity of primate APOBEC3C
The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Multiple integrated analyses determined why rhA3C was not active against HIV-1 and how to increase this activity. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity also promoted dimerization. Although rhA3C shares a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C, establish the amino acid network for dimerization and increased activity, and track the loss and gain of A3C antiviral activity in primates. The coevolutionary analysis of the A3C dimerization interface provides a basis from which to analyze dimerization interfaces of other A3 family members.
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- Award ID(s):
- 1757946
- PAR ID:
- 10292209
- Date Published:
- Journal Name:
- bioRxiv
- ISSN:
- 2692-8205
- 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/10.1101/2021.07.13.452235
