Effects of bulky polycyclic aromatic hydrocarbon adducts on DNA replication by exonuclease-deficient T7 and T4 DNA polymerases.


In vitro DNA replication by exonuclease-deficient T7 DNA polymerase (Sequenase) and an exonuclease deficient T4 DNA polymerase was examined on a 244-nucleotide DNA template treated with three electrophilic polycyclic aromatic hydrocarbon (PAH) metabolites: racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FADE), or 3,4-epoxy-3,4-dihydrocyclopenta[cd]pyrene (CPPE). The DNA replication terminated opposite template guanines and, to a lesser extent, at template adenines, as expected, as purines were modified preferentially by the chemical treatments. Analysis of the products synthesized on the damaged templates indicated that bypass replication by Sequenase proceeded in three steps: (1) replication first terminated one base 3' to each adduct; (2) a nucleotide was then incorporated opposite the PAH-modified base; and (3) replication continued at some sites to give full bypass of the lesions. The rate of lesion bypass was affected by the type of chemical adduct, the sequence context of the adduct, and the concentration of deoxynucleoside triphosphates. Short DNA repeats appeared to facilitate translesion replication.



Nucleotide Analogs / Template Lesions, Nucleotide Incorporation

One line summary:

Elucidated bypass replication mechanism by Sequenase.


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