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The N 2 -Ethylguanine and the O 6 -Ethyl- and O 6 -Methylguanine Lesions in DNA: Contrasting Responses from the “Bypass” DNA Polymerase η and the Replicative DNA Polymerase α

2003; American Chemical Society; Volume: 16; Issue: 12 Linguagem: Inglês

10.1021/tx034164f

1520-5010

Fred W. Perrino, Patrick Blans, Scott Harvey, Stacy L. Gelhaus, Colleen E. McGrath, Steven A. Akman, G. Scott Jenkins, William R. LaCourse, James C. Fishbein,

Advanced biosensing and bioanalysis techniques

The effects of N2-ethylGua, O6-ethylGua, and O6-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases α and η have been investigated. The N2-ethylGua adduct blocks polymerization by the replicative DNA polymerase α to a much greater extent than does the O6-ethyl- or the O6-methylGua adducts. The DNA polymerase η efficiently and accurately bypasses the N2-ethylGua lesion but like DNA polymerase α is similarly blocked by the O6-ethyl- or the O6-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N2-ethylGua and the O6-ethylGua adducts by the DNA polymerases α and η and extension from 3'-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N2-ethylGua adduct by DNA polymerase α is approximately 104-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N2-ethylGua:Cyt 3'-terminus by DNA polymerase α is approximately 103-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N2-ethylGua lesion by the DNA polymerase η is about 370-fold more efficient than by the DNA polymerase α, and extension from the N2-ethylGua:Cyt 3'-terminus by the DNA polymerase η is about 3-fold more efficient than by the DNA polymerase α. Furthermore, the DNA polymerase η preferably inserts the correct nucleotide Cyt opposite the N2-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O6-ethylGua adduct by the DNA polymerases α and η. In reactions containing both DNA polymerases α and η, synthesis past the N2-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N2-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase α and lesion bypass catalyzed by DNA polymerase η.