Replication, Repair and Translesion Polymerase Bypass of N6-Oxopropenyl-2'-deoxyadenosine.

Abstract:

The oxidative stress products malondialdehyde and base propenal react with DNA bases forming the adduction products 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) and N6-(oxypropenyl)-2'-deoxyadenosine (OPdA). M1dG is mutagenic in vivo and miscodes in vitro, but little work has been done on OPdA. To further our understanding of the effect of OPdA on polymerase activity and mutagenicity, we evaluated the ability of the translesion DNA polymerases hPols η, κ, and ι to bypass OPdA in vitro. hPols η and κ inserted dNTPs opposite the lesion and extended the OPdA-modified primer to the terminus. hPol ι inserted dNTPs opposite OPdA but failed to fully extend the primer. Steady-state kinetic analysis indicated that these polymerases preferentially insert dTTP opposite OPdA, although less efficiently than opposite dA. Minimal incorrect base insertion was observed for all polymerases, and dCTP was the primary mis-insertion event. Examining replicative and repair polymerases revealed little effect of OPdA on the Sulfolobus solfataricus polymerase Dpo1 or the Klenow fragment of Escherichia coli DNA polymerase I. Bacteriophage T7 DNA polymerase displayed reduced OPdA bypass compared to unmodified DNA, and OPdA nearly completely blocked the activity of base excision repair polymerase hPol β. This work demonstrates that bypass of OPdA is generally error-free, modestly decreases the catalytic activity of most polymerases, and blocks hPol β polymerase activity. Although mis-insertion opposite OPdA is relatively weak, the efficiency of bypass may introduce A → G transitions observed in vivo.

Polymerases:

Topics:

Nucleotide Analogs / Template Lesions, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol beta Maddukuri L2013 Template lesions Stalls Reaction: Nucleotide incorporation; Substrate: n/a; DNA lesion: OPdA
Human Pol kappa Maddukuri L2013 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dTTP; DNA lesion: OPdA
Human Pol eta Maddukuri L2013 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dTTP; DNA lesion: OPdA
Human Pol iota Maddukuri L2013 Template lesions Idles Reaction: Nucleotide incorporation; Substrate: dTTP; DNA lesion: OPdA
SsoDpo1 Maddukuri L2013 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dTTP; DNA lesion: OPdA
T7 Maddukuri L2013 Template lesions Idles Reaction: Nucleotide incorporation; Substrate: dTTP; DNA lesion: OPdA

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