Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis

Highlights

• Archaeal GO glycosylase (AGOG) is a major 8-oxo-dG repair enzyme in the hyperthermophilic archaeon Thermococcus kodakarensis.

• AGOG is non-essential for viability in Thermococcus kodakarensis suggesting redundant pathways for oxidative DNA damage repair.

• TkoEndoIV is essential for viability in Thermococcus kodakarensis and is important for 8-oxo-dG repair and base excision repair.

• Archaeal 8-oxo-dG repair was reconstituted in vitro using AGOG, EndoIV, DNA polymerase, Fen1 and DNA ligase.

Abstract

Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.