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Replicative Bypass Studies of α-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro
Nicole L Williams1, Nicholas J Amato1, Yinsheng Wang1
1Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , 501 Big Springs Road, Riverside, California 92521-0403, United States.
Abstract:
Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 104-105 DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give α-anomeric lesions. In this study, we assessed the replicative bypass of α-dA, α-dT, α-dC, and α-dG in template DNA by conducting primer extension assays with the use of purified translesion synthesis DNA polymerases. Our results revealed that human polymerase (Pol) η, but not human Pol κ, Pol ι, or yeast Pol ζ, was capable of bypassing all of the α-dN lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η was the most efficient in inserting the correct nucleotides opposite the modified nucleosides, with the relative efficiencies of nucleotide incorporation following the order of α-dA > α-dG > α-dT > α-dC. Additionally, human Pol η was found to misincorporate dTMP opposite α-dT and dCMP opposite α-dC at frequencies of 66% and 24%, respectively, whereas α-dA and α-dG were weakly miscoding. These findings provided important knowledge about the effects these α-dN lesions have on the fidelity and efficiency of DNA replication mediated by human Pol η.
Insights
Human DNA polymerase eta (Pol η) efficiently bypasses challenging α-anomeric DNA lesions, crucial for maintaining genomic integrity during replication. This polymerase demonstrates high fidelity in nucleotide incorporation opposite these lesions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Genomic integrity is vital and constantly threatened by DNA damaging agents.
- Reactive oxygen species (ROS) are significant endogenous DNA damaging agents.
- Hydroxyl radicals can cause α-anomeric DNA lesions, impacting DNA replication.
Purpose of the Study:
- To investigate the replicative bypass of α-anomeric DNA lesions (α-dA, α-dT, α-dC, α-dG).
- To determine the efficiency and fidelity of various translesion synthesis DNA polymerases in bypassing these lesions.
Main Methods:
- Primer extension assays using purified translesion synthesis DNA polymerases.
- Steady-state kinetic measurements to assess nucleotide incorporation efficiencies.
- Analysis of misincorporation frequencies opposite α-anomeric lesions.
Main Results:
- Human polymerase eta (Pol η) uniquely bypassed all tested α-anomeric lesions (α-dA, α-dT, α-dC, α-dG).
- Pol η showed the highest efficiency in inserting correct nucleotides, with order α-dA > α-dG > α-dT > α-dC.
- Pol η exhibited misincorporation of dTMP opposite α-dT (66%) and dCMP opposite α-dC (24%).
Conclusions:
- Human Pol η plays a critical role in replicating DNA containing α-anomeric lesions.
- Pol η's efficiency and fidelity are key to mitigating the mutagenic potential of these lesions.
- Understanding Pol η's mechanism provides insights into DNA repair and genomic stability.