The Kinetic Analysis Of Bypassing 8-oxoG DNA Damage By Yeast Polymerase ?_core

Reactive oxygen species damage DNA bases to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine?8-oxoG?,which results in G:C to T:A transversions.To better understand mechanisms of dNTP incorporation opposite 8-oxoG,we performed pre-steady-state kinetic analysis of nucleotide incorporation using the catalytic core of yeast DNA polymerase?(Pol?_core,residues 1-513)instead of full-length Pol?,eliminating potential effects of the C-terminal C₂H₂ sequence motif on dNTP incorporation.Kinetic analysis showed that Pol?_core preferred to incorporate dCTP opposite 8-oxoG.A lack of a pre-steady-state kinetic burst for Pol?_core suggested that dCTP incorporation is slower than the dissociation of the polymerase from DNA.The extension products beyond the 8-oxoG were determined by LC-MS/MS and showed that 57%of the products corresponded to the correct incorporation?C?and 43%corresponded to dATP misincorporation.More dATP was incorporated opposite8-oxoG with a mixture of dNTPs than predicted using only a single dNTP.The kinetic analysis of 8-oxoG bypass by yeast DNA Pol?_core provides further understanding of the mechanism of mutation at this oxidation lesion with yeast DNA polymerase?.