Duarte V, Muller J G, Burrows C J
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA.
Nucleic Acids Res. 1999 Jan 15;27(2):496-502. doi: 10.1093/nar/27.2.496.
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-.
DNA碱基的氧化损伤通常会导致氧化嘌呤的形成，尤其是7,8 - 二氢 - 8 - 氧代鸟嘌呤（8 - oxoG）和7,8 - 二氢 - 8 - 氧代腺嘌呤（8 - oxoA），前者是一种众所周知的诱变损伤。由于与正常碱基相比，8 - oxoG很容易进一步氧化，因此在体外研究了DNA合成过程中与8 - oxoG的氧化形式相对应位置的碱基插入情况。首先用不同的单电子氧化剂处理含有单个8 - oxoG损伤的合成模板，或在单线态氧条件下处理，然后进行由Klenow片段外切酶（Kf exo -）、小牛胸腺DNA聚合酶α（pol α）或人DNA聚合酶β（pol β）催化的引物延伸反应。与先前的报道一致，所有三种DNA聚合酶在8 - oxoG相对位置上均选择性地插入dAMP和dCMP。有趣的是，发现8 - oxoG的氧化会导致Kf exo - 在损伤相对位置插入dAMP和dGMP，并在修饰碱基位点处短暂抑制引物延伸。此外，该损伤在pol α和pol β进行DNA合成时构成一个障碍。对含有8 - oxoA修饰的模板寡核苷酸的实验表明，8 - oxoA及其氧化形式均可指导Kf exo - 直接插入dTMP。用IrCl62 - 氧化前后含8 - oxoG的寡核苷酸的质谱分析结果与主要是8 - oxoG位点的氧化情况一致，主要产物是胍基乙内酰脲部分的形成。未获得形成无碱基位点的证据。这些结果表明，8 - oxoG的一种氧化形式，可能是胍基乙内酰脲，可能在DNA合成过程中指导dNTP的错读和错配插入。如果这样的过程发生在体内，它将代表一种点诱变损伤，导致G→T和G→C颠换。然而，8 - oxoA的相应氧化形式在Kf exo - 进行DNA合成过程中主要表现为T的正确插入。
