Abasic Translesion Synthesis by DNA Polymerase β Violates the “A-rule”

The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo. DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule. However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes. Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion. Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base. Pol alpha strongly favors incorporation of dAMP directly opposite the lesion. In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion. In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion. Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site. Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors. In contrast, pol alpha shows no propensity for this type of synthesis. The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.