MECHANISM OF BYPASS SYNTHESIS THROUGH AN ABASIC SITE ANALOG BY DNA-POLYMERASE-I

Bypass synthesis by DNA polymerase I was studied using synthetic 40-nu cleotide-long gapped duplex DNAs each containing a site-specific abasi c site analog, as a model system for mutagenesis associated with DNA l esions. Bypass synthesis proceeded in two general stages: a fast polym erization stage that terminated opposite the abasic site analog, follo wed by a slow bypass stage and polymerization down to the end of the t emplate. The position of the 3'-terminus of the primer relative to the abasic site analog did not affect bypass synthesis in the range of -1 to -5. In contrast, bypass synthesis increased with the distance of t he 5'-boundary of the gap from the lesion for up to 3-fold in the rang e of +1 to +9. Bypass synthesis was severely inhibited by moderate con centrations of salts, and under conditions that were optimal for the s ynthetic activity of DNA polymerase I (100 mM K+), bypass synthesis wa s completely inhibited (<0.02% bypass). Elimination of the 3'-->5' pro ofreading exonuclease activity of the polymerase, by using a mutant DN A polymerase, caused a dramatic 10-60-fold increase in bypass synthesi s. Determination of the kinetic parameters for insertion opposite the abasic site analog revealed a strong preference for the insertion of d AMP, dictated by a lower K-m and a higher k(cat) as compared to the ot her nucleotides. The rate of bypass was increased by omitting one or t wo dNTPs, most likely due to the facilitation of the polymerization pa st the lesion.