ABASIC TRANSLESION SYNTHESIS BY DNA-POLYMERASE-BETA VIOLATES THE A-RULE - NOVEL TYPES OF NUCLEOTIDE INCORPORATION BY HUMAN DNA-POLYMERASE-BETA AT AN ABASIC LESION IN DIFFERENT SEQUENCE CONTEXTS

The ''A-rule'' reflects the preferred incorporation of dAMP opposite a basic 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 ab asic lesions in eucaryotes. Purified human DNA polymerases beta and al pha 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. Extens ion past the lesion is measured in 4 sequence contexts, varying the do wnstream template base. Pol alpha strongly favors incorporation of dAM P 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 a nalyze misalignment incorporation directed by a template base downstre am from the lesion, Lesion bypass by pol beta occurs predominantly by ''skipping over'' the lesion, by insertion of a nucleotide complementa ry to an adjacent downstream template site. Misalignment incorporation for pol beta occurs by a novel ''dNTP-stabilized'' mechanism resultin g in both deletion and base substitution errors. In contrast, pol alph a shows no propensity for this type of synthesis, The misaligned DNA s tructures generated during dNTP-stabilized lesion bypass do not confor m to misaligned structures reported previously.