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Title page for ETD etd-10082007-083404

Type of Document Dissertation
Author Christov, Plamen Petkov
URN etd-10082007-083404
Title Synthesis and in vitro replicatuion studies of N5-alkylated formamidopyrimicine (FAPy-dGuo) adducts in DNA.
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Carmelo Rizzo Committee Chair
Brian Bachmann Committee Member
Frederick Guengerich Committee Member
Michael Stone Committee Member
Ned Porter Committee Member
  • vinyl chloride
  • 2-oxoethyl-FAPy-dGuo
  • DNA damage
  • MeFAPy-dGuo
  • in vitro replication
  • DNA adducts
  • Pyrimidines -- Synthesis
  • Nucleosides -- Synthesis
  • Vinyl chloride -- Carcinogenicity
Date of Defense 2007-10-03
Availability unrestricted
Methylating agents react with DNA to give the cationic N7-methyl deoxyguanosine adduct, which can undergo hydrolytic opening of the imidazole ring to form 2,6-diamino-4-hydroxy-N5-(methyl)-formamido-pyrimidine (MeFAPy-dGuo) adduct. MeFAPy-dGuo lesion represents an important but largely unexplored class of DNA lesions, because of the lack of feasible ways for the preparation of oligonucleotides containing FAPy adducts. We have synthesized a MeFAPy-dGuo phosphoroamidite for a first time. This phosphoroamidite was synthesized in four steps and 25% overall yield and was used for the preparation of oligonucleotides containing the MeFAPy-dGuo lesion. The in vitro bypass and full length-extension of the MeFAPy-dGuo adduct was examined with prokaryotic and eukaryotic DNA polymerases. The MeFAPy-dGuo adduct was found to be highly miscoding; in addition to the correct insertion of dCTP opposite the lesion all three prokaryotic DNA polymerases inserted dATP, dGTP and dTTP. The MeFAPy-dGuo adduct was also found to be a complete block to replication catalyzed by human replicative DNA polymerase pol delta and highly miscoding to the human DNA polymerases pols kapa, eta, and iota, and the combinations of human pols kapa/iota and pols eta/iota. The sequences of the full-length extension products were determined using a new LC-ESI/MS/MS strategy. The use of biotinated primers improved the sensitivity of the LC-ESI/MS/MS method, and an oligonucleotide internal standard was used to quantitate the full-length extension products.

Vinyl chloride is an important industrial monomer and an established human carcinogen. The carcinogenic species is 2-chlorooxirane, which arises from in vivo oxidation of vinyl chloride. The major adduct form the reaction of 2-chlorooxirane with deoxyguanosine is the cationic N7-(2-oxoethyl) adduct; however, this species has been shown to be non-mutagenic. We hypothesized that the corresponding (2-oxoethyl)-FAPy-dGuo lesion may be formed and could contribute to the mutagenicity of vinyl chloride and related species. To identify the formation of the (2-oxoethyl)-FAPy-dGuo lesion, an independent synthesis was developed to provide an authentic sample of the corresponding FAPy-Guo. When deoxyguanosine and oligonucleotide were treated with 2-acetoxyoxirane, a surrogate for 2-chlorooxirane, followed by 0.1 M NaOH, the (2-oxoethyl)-FAPy-dGuo lesion was detected.

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