Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.
Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.
Duarte
V
V
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA.
Muller
J G
JG
Burrows
C J
CJ
D000818:Animals; D002417:Cattle; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D006827:Hydantoins; D007495:Iridium; D016296:Mutagenesis; D009532:Nickel; D016877:Oxidants; D010084:Oxidation-Reduction
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D006827:Hydantoins; D016877:Oxidants; D006151:Guanosine; C010978:hexachloroiridic acid; C046215:8-hydroxyguanosine; D007495:Iridium; C012873:2'-deoxy-5'-adenosine monophosphate; C007257:2'-deoxyguanosine 5'-phosphate; D009532:Nickel; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase