RESUMEN
Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+â¢) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+⢠to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.
Asunto(s)
Daño del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Desoxicitidina/química , Radicales Libres/química , ADN/química , Desoxicitidina/análogos & derivados , Desoxicitidina/efectos de la radiación , Desoxiguanosina/química , Oximas/química , Oximas/efectos de la radiación , Fotólisis , Rayos UltravioletaRESUMEN
DNA polymerase θ (Pol θ) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol θ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol θ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4'-oxidation (pC4-AP). Covalent modification of Pol θ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol θ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol θ and suggests a path forward for designing inhibitors of this attractive anticancer target.