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DNA synthesis arrest at C4'-modified deoxyribose residues
M T Hess1, U Schwitter, M Petretta
1Institute of Pharmacology and Toxicology, University of Zürich-Tierspital, Switzerland.
Abstract:
Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives. A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases. Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack. We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct. Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested. These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties. However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates. The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.
Insights
DNA synthesis halts when encountering damaged DNA templates. Altered C4' deoxyribose structures, like inversions or adducts, block DNA polymerase progression, impacting replication fidelity and suggesting therapeutic targets.
Area of Science:
- Molecular Biology
- Biochemistry
- Genotoxicology
Background:
- Genotoxic agents often create DNA base lesions that impede DNA polymerase activity.
- Understanding DNA synthesis termination on damaged templates is crucial for DNA repair and replication studies.
Purpose of the Study:
- To investigate the mechanism of DNA synthesis termination on templates with modified C4' deoxyribose configurations.
- To assess the impact of C4' deoxyribose variants on DNA polymerase progression and base-pairing fidelity.
Main Methods:
- Utilized the Klenow fragment of Escherichia coli DNA polymerase I as a model enzyme.
- Synthesized DNA templates containing C4' deoxyribose inversion or specific adducts (selenophenyl, pivaloyl).
- Performed primer extension assays with varying deoxyribonucleotides to analyze polymerase activity and fidelity.
Main Results:
- DNA synthesis was transiently arrested at C4' inversion and significantly blocked by C4' adducts.
- Termination sites were precisely mapped relative to the C4' adducts.
- Base-pairing fidelity remained intact across all C4' variants, even with a proofreading-deficient polymerase mutant.
Conclusions:
- Altered C4' deoxyribose moieties maintain template coding ability but impede DNA polymerase progression.
- Backbone distortion and steric hindrance are key factors in DNA synthesis arrest on damaged templates.
- C4' adducts represent potential targets for novel chemotherapeutic strategies.