Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by the catalytic core of yeast DNA polymerase η

Abstract

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 η (Pol ηcore, residues 1-513) instead of full-length Pol η, eliminating potential effects of the C-terminal C2H2 sequence motif on dNTP incorporation. Kinetic analysis showed that Pol ηcore preferred to incorporate dCTP opposite 8-oxoG. A lack of a pre-steady-state kinetic burst for Pol ηcore 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. The kinetic analysis of 8-oxoG bypass by yeast DNA Pol ηcore, provides further understanding of the mechanism of mutation at this oxidation lesion with yeast DNA polymerase η.

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

“…In the case of reverse transcription, it is a combination of the rate of catalysis (condensation between 3′ end of the primer strand and a triphosphate being inserted opposite a studied base) and the rate of dissociation between the primer-template complex and the enzyme after the primer extension. For polymerases, k cat is normally defined by the rate of the slower dissociation step, 62–64, 81, 82 thus it was not surprising that we observed very close values with overlapping confidence intervals for formation of OG-A (2.5 ± 0.1 min −1 ), OG-C (2.4 ± 0.2 min −1 ), and G-C (2.7 ± 0.2 min −1 ) base pairs. Similarities between turnover numbers for incorporation of a base opposite G and OG have been previously reported for translesion DNA polymerase η and HIV-1 RT by the Guengerich laboratory.…”

“…Similarities between turnover numbers for incorporation of a base opposite G and OG have been previously reported for translesion DNA polymerase η and HIV-1 RT by the Guengerich laboratory. 62, 64 Unlike OG, the rate of phosphodiester bond formation apparently was affected by the presence of hydantoin lesions in the template strongly enough to result in lower k cat values for Gh-A (0.69 ± 0.08 min −1 ), Gh-G (0.55 ± 0.03 min −1 ), S -Sp-A (0.071 ± 0.006 min −1 ), S -Sp-G (0.056 ± 0.002 min −1 ), R -Sp-A (0.196 ± 0.003 min −1 ), and R -Sp-G (0.12 ± 0.01 min −1 ) base pairs. The k cat values for templates containing different products of guanine oxidation follow the same rule as reactivities determined for the nucleotide insertion assays: OG>Gh> R -Sp> S -Sp (Figure 2 and Table S1).…”

“…Considering that Sp does not form stable base pairs with any natural nucleotides, 61 insertion of comparable amounts of A and G opposite the hydantoin most likely can be attributed to fitting in the SuperScript III active site due to a similar size and minor groove binding pattern of Sp to C or T, 86–88 rather than to efficient hydrogen-bonding with A or G. The ratio of A and C incorporated opposite OG by the polymerase can significantly deviate from the one calculated based on kinetic parameters in the presence of multiple triphosphates. 64 Thus we tested whether this was true for SuperScript III using a gel assay (Figure S7 and S8). Since only qualitative characterization of the insertion rate was possible we attempted to adjust concentrations to achieve close to a 1:1 insertion ratio and compared it to the one expected from the reaction rates calculated at those concentrations (Table S3).…”

“…Insertion of A or C opposite OG and A or G opposite the hydantoin lesions was observed that correlated with the previous reports for DNA containing these bases. 37, 61, 64 We also report steady-state kinetic parameters for single nucleotide insertion opposite the studied lesions in the 5′-AXC-3′ sequence context by the SuperScript III reverse transcriptase. Results of this assay indicate a preference for insertion of A opposite OG and G opposite the Gh and Sp diastereomers.…”

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass

“…[4] HumanD NA polymerase k (Pol k)p refers to insert dATP rather than dCTP opposite 8-oxoG. [3] O 6 -MeG is am utagenic DNA lesion, arising from the methylation of guanine( G). [3] O 6 -MeG is am utagenic DNA lesion, arising from the methylation of guanine( G).…”