DNA-SYNTHESIS ARREST AT C4'-MODIFIED DEOXYRIBOSE RESIDUES

Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defe ctive templates, we tested the capacity of a model enzyme (Klenow frag ment of Escherichia call DNA polymerase I) to catalyze primer elongati on across a series of C4' deoxyribose derivatives. A site with inverte d C4' configuration or two different C4' deoxyribose adducts were intr oduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases, Inverted deoxyribose moieties ma y arise in cellular DNA as a product of C4' radical attack. We found t hat DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose addu cts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pival oyl adduct. Primer extension studies in the presence of single deoxyri bonucleotides showed intact base pairing fidelity opposite all three C 4' variants regardless of whether the Klenow fragment or its proofread ing-deficient mutant was tested. These results imply that the coding a bility of template bases is maintained at altered C4' deoxyribose moie ties. However, their capacity to impede DNA polymerase progression ind icates that backbone distortion and steric hindrance are important det erminants of DNA synthesis arrest on damaged templates. The strong inh ibition by C4' adducts suggests a potential target for new chemotherap eutic strategies.