Kinetic basis for the differing response to an oxidative lesion by a replicative and a lesion bypass DNA polymerase...

8-Oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), a major oxidative DNA lesion, exhibits ambiguous coding potential and can lead to genomic mutations. Tight control of 8-oxoG bypass during DNA replication is therefore extremely important in hyperthermophiles as the rate of oxidative damage to DNA is significantly increased at high temperatures. Here we employed pre-steady state kinetics to compare the kinetic responses to an 8-oxoG lesion of the main replicative and lesion bypass DNA polymerases of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon. Upon encountering 8-oxoG, PolB1, the replicative DNA polymerase, was completely stalled by the lesion, as its 3' → 5' exonuclease activity increased significantly and outcompeted its slowed polymerase activity at and near the lesion site. In contrast, our results show that Dpo4, the lone Y-family DNA polymerase in S. solfataricus, can faithfully and efficiently incorporate nucleotides opposite 8-oxoG and extend from an 8-oxoG:C base pair with a mechanism similar to that observed for the replication of undamaged DNA. Furthermore, we show that the stalling of PolB1 at the lesion site can be relieved by Dpo4. Finally, the 3' → 5' exonuclease activity of PolB1 was the highest when 8-oxoG was mispaired with an incorrect nucleotide and could therefore correct rare mistakes made by Dpo4 during 8-oxoG bypass. These results provide a kinetic basis for a potential polymerase switching mechanism during 8-oxoG bypass whereby Dpo4 can switch with the stalled PolB1 at the replication fork to bypass and extend the damaged DNA and then switch off of the DNA substrate to allow continued replication of undamaged DNA by the more faithful PolB1.