Department of Biology, Emory University, Atlanta, GA, USA.
PLoS Genet. 2013;9(8):e1003682. doi: 10.1371/journal.pgen.1003682. Epub 2013 Aug 1.
8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.
8-氧鸟嘌呤是由氧化损伤引起的最常见和最具突变性的 DNA 碱基损伤之一。然而，一直无法在体内研究已知的 8-氧鸟嘌呤碱基的复制，以确定其复制的准确性、各种因素对该准确性的影响，以及 8-氧鸟嘌呤可能对复制造成的障碍程度。我们已经能够使用单链寡核苷酸转化将单个 8-氧鸟嘌呤置于酿酒酵母染色体的特定位置，并在完全正常的染色体背景下研究其复制。在复制过程中，8-氧鸟嘌呤被识别为损伤，并触发 Pol η 进行跨损伤合成的转换，Pol η 以约 94%的准确性（在 8-氧鸟嘌呤的对面插入一个 C）复制 8-氧鸟嘌呤。在没有 Pol η 的情况下，至少有三分之一的时间观察到模板切换到新合成的姐妹染色单体；在没有 Pol η 的情况下，8-氧鸟嘌呤的复制准确性小于 40%。错配修复（MMR）系统在 8-氧鸟嘌呤复制中发挥重要作用。MMR 会阻止模板切换，即使在没有 Pol η 的情况下，当 MMR 活跃时，复制准确性也约为 95%。这些发现表明，鉴于细胞中避免 8-氧鸟嘌呤复制错误的重叠机制，8-氧鸟嘌呤的诱变威胁更多地归因于其丰度，而不是单个损伤的影响。此外，此处使用的方法应该适用于任何可以稳定掺入合成寡核苷酸的损伤的研究。
