In Vivo Bypass of 8-oxodG

Abstract

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.

Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

“…Overlaps between MMR and TLS were confirmed by a recent in vivo study in yeast. The A residues opposite 8-oxoG were removed by MMR, subsequently triggering re-replication and, in the absence of MMR, by pol TLS, with an accuracy of insertion of dCTP opposite 8-oxoG of 94% (37).…”

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

“…pol accurately and efficiently incorporates nucleotides opposite UV-induced cyclobutane pyrimidine dimers (24,25). In addition, pols and are involved in error-prone and errorfree TLS, respectively, of 8-oxo-dG in vivo; pol knockdown reduces G to T transversion mutations caused by 8-oxo-dG in human cells (26), and the absence of pol decreases the accuracy of TLS across 8-oxo-dG in yeast (27).…”

Trinucleotide expansion in disease: why is there a length threshold?

“…Strand displacement during long patch BER is around the same size or larger when CAG TNRs are the repair substrate [ 15,16 ]. Moreover, small chemical lesions such as 8-oxo-guanine can trigger a switch to translesion synthesis by Pol η in yeast [ 17 ]. Polymerase pausing is noted in long non-coding TNRs, and the size of the loops formed during fork reversal [ 18 ] or strand-switching [ 19 ] mechanisms have the potential to promote even larger loops.…”