Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I.


Bypass synthesis by DNA polymerase I was studied using synthetic ...
Bypass synthesis by DNA polymerase I was studied using synthetic 40-nucleotide-long gapped duplex DNAs each containing a site-specific abasic site analog, as a model system for mutagenesis associated with DNA lesions. Bypass synthesis proceeded in two general stages: a fast polymerization stage that terminated opposite the abasic site analog, followed by a slow bypass stage and polymerization down to the end of the template. The position of the 3'-terminus of the primer relative to the absic site analog did not affect bypass synthesis in the range of -1 to -5. In contrast, bypass synthesis increased with the distance of the 5'-boundary of the gap from the lesion for up to 3-fold in the range of +1 to +9. Bypass synthesis was severely inhibited by moderate concentrations of salts, and under conditions that were optimal for the synthetic activity of DNA polymerase I (100 mM K+), bypass synthesis was completely inhibited (< 0.02% bypass). Elimination of the 3'-->5' proofreading exonuclease activity of the polymerase, by using a mutant DNA polymerase, caused a dramatic 10-60-fold increase in bypass synthesis. Determination of the kinetic parameters for insertion opposite the abasic site analog revealed a strong preference for the insertion of dAMP, dictated by a lower Km and a higher kcat as compared to the other nucleotides. The rate of bypass was increased by omitting one or two dNTPs, most likely due to the facilitation of the polymerization past the lesion.



Nucleotide Analogs / Template Lesions, Nucleotide Incorporation


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