Ribonucleotide incorporation by human specialized DNA polymerases opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) lesion.

DNA polymerases (Pols) are specialized enzymes devoted to DNA synthesis. However, in spite of their specificity for dNTPs, they can incorporate ribonucleotides (rNTPs) during DNA replication. Ribonucleotides are a source of genomic instability since they could lead to spontaneous strand breaks and stalling of Pols at replication forks. Ribonucleotides can be inserted in the genome also by reparative polymerases belonging to X family, such as Pols beta and lambda.These Pols have essential functions in Base Excision Repair (BER), Non-Homologous End Joining (NHEJ) pathways, as well as in specialized form of translesion synthesis (TLS) of oxidative lesions. In particular, they are involved in bypassyng 7,8-dihydro-8-oxoguanine (8-oxo-G), the most abundant oxidative DNA damage, estimated in 1,000 - 10,000 8-oxo-G damaged bases generated spontaneously in every cell per day. However, it is not known so far whether these Pols can also incorporate rNTPs opposite this lesion. Therefore, we compared ribonucleotide incorporation catalyzed by Pols beta and lambda opposite normal or damaged bases under physiological concentrations of metal activators and nucleotides. We found that both Pol beta and Pol lambda could bypass the 8-oxo-G lesion by incorporating rCTP or rATP, although with different efficiencies, and that their fidelity was influenced by the structure of the DNA template. Our in vitro kinetic data suggested that Pol beta, more than Pol lambda, could be a source of rNTPs incorporation in the genome, especially after oxidative stress.