Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP to be inserted opposite 7,8-dihydro-8-oxoguanine .

Accurate copying of the genome by DNA polymerases is challenging due in part to the continuous damage inflicted on DNA, which results from its contact with reactive oxygen species (ROS), producing lesions such as 7,8-dihydro-8-oxoguanine (8-oxoG). The deleterious effects of 8-oxoG can be attributed to its dual coding potential that leads to G → T transversions. The wild-type (wt) pol α family DNA polymerase from bacteriophage RB69 (RB69pol) prefers to insert dCMP as opposed to dAMP when situated opposite 8-oxoG by >2 orders of magnitude as demonstrated using pre-steady-state kinetics (kpol/Kd,app). In contrast, the Y567A mutant of RB69pol inserts both dCMP and dAMP opposite 8-oxoG rapidly and with equal efficiency. We have determined the structures of preinsertion complexes for the Y567A mutant with dATP and dCTP opposite a templating 8-oxoG in a 13/18mer primer-template (P/T) at resolutions of 2.3 and 2.1 A, respectively. Our structures show that the 8-oxoG residue is in the anti conformation when paired...