In vivo bypass of 8-oxodG.
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.
Department of Biology, Emory University, Atlanta, GA, USA.
Gina P
GP
Rodriguez
Joseph B
JB
Song
Gray F
GF
Crouse
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000235:genetics; D006147:Guanine / Q000378:metabolism; D016296:Mutagenesis; D018384:Oxidative Stress / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000099270:Y-Family DNA Polymerases
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D006147:Guanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases