Bypass of N²-ethylguanine by human DNA polymerase κ.

The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N(2)-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3' termini opposite N(2)-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N(2)-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn(2+) compared to Mg(2+).

Lesion bypass of N2-ethylguanine by human DNA polymerase iota.

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+.

Replication of N2-ethyldeoxyguanosine DNA adducts in the human embryonic kidney cell line 293.

N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX.

Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli.

Exocyclic alkylamino purine adducts, including N(2)-ethyldeoxyguanosine, N(2)-isopropyldeoxyguanosine, and N(6)-isopropyldeoxyadenosine, occur as a consequence of reactions of DNA with toxins such as the ethanol metabolite acetaldehyde, diisopropylnitrosamine, and diisopropyltriazene. However, there are few data addressing the biological consequences of these adducts when present in DNA. Therefore, we assessed the mutagenicities of these single, chemically synthesized exocyclic amino adducts when placed site-specifically in the supF gene in the reporter plasmid pLSX and replicated in Escherichia coli, comparing the mutagenic potential of these exocyclic amino adducts to that of O(6)-ethyldeoxyguanosine.

The structure of DNA dictates purine atom site selectivity in alkylation by primary diazonium ions.

The 1-propanediazonium ion, generated from N'-nitro-N-nitroso-N-propylguanidine in aqueous solutions, was reacted with the purine nucleosides dGuo and dAdo or single-stranded or double-stranded DNA. After nucleobase liberation by acid hydrolysis, the percent yields of products were determined by LC/MS using either isotopically distinct internal standards in the case of the nucleoside reactions or an internal standard and the ratios of response factors of all other products that were separately determined in the case of the reactions with DNA. In the reactions of nucleosides, products of both n-propylation and iso-propylation at all of the heroatoms were observed.

Polymerization past the N2-isopropylguanine and the N6-isopropyladenine DNA lesions with the translesion synthesis DNA polymerases eta and iota and the replicative DNA polymerase alpha.

The effects of N2-isopropylGua and N6-isopropylAde adducts in template DNA on polymerization by the human replicative DNA polymerase alpha (B-family) and the translesion synthesis DNA polymerases eta and iota (Y-family) were investigated. A direct comparison between the accuracies of DNA synthesis using catalytic fragments of the human DNA polymerases eta and iota is reported. We show that the N2-isopropylGua adduct is a powerful block to polymerization by DNA polymerase alpha.

Determinants of selectivity in alkylation of nucleosides and DNA by secondary diazonium ions: evidence for, and consequences of, a preassociation mechanism.

Reactions have been carried out in which 1,3-diisopropyltriazene or N-isopropyl-N-(1-hydroxyethyl)nitrosamine has been decomposed in neutral, buffered aqueous media in the presence of (15N2)2'-deoxyguanosine and (15N6)2'-deoxyadenosine. The products of covalent attachment of the isopropyl cation, derived from the isopropyl diazonium ion, to the heteroatoms of the purines have been separated and quantified by HPLC/electrospray mass spectrometry by employing isotopically distinct synthetic standards. The results indicate that the two different precursors of the isopropyl cation result in the formation of different yields of products in the reactions at all of the heteroatoms of both purines, outside experimental error, except possibly in the case of the N3 position of dAdo.

The N2-ethylguanine and the O6-ethyl- and O6-methylguanine lesions in DNA: contrasting responses from the "bypass" DNA polymerase eta and the replicative DNA polymerase alpha.

The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts.

Thiolytic chemistry of alternative precursors to the major metabolite of the cancer chemopreventive oltipraz.

The compounds 7-methyl-6,8-bis(methyldisulfanyl)pyrrolo[1,2-a]pyrazine (5; "bis disulfide") and methanethiosulfonic acid S-((6-(methanesulfonylsulfanyl)-7-methyl)pyrrolo[1,2-a]pyrazin-8-yl) ester (6; "bis methanesulfonic acid thioester") have been synthesized to serve as alternative precursors to the major metabolite, 4, of the cancer chemopreventive oltipraz, 1, to test whether they possess similar biological activities. In the present work the mechanisms by which these compounds react with glutathione have been investigated in order to validate the assumption that they would be chemically competent in the presence of the biological thiols to give the oltipraz metabolite. A kinetic and product study was carried out in mainly aqueous media, View Article and Find Full Text PDF