The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N(2)-ethylGua and the O(6)-ethylGua adducts by the DNA polymerases alpha and eta 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 N(2)-ethylGua adduct by DNA polymerase alpha is approximately 10(4)-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N(2)-ethylGua:Cyt 3'-terminus by DNA polymerase alpha is approximately 10(3)-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N(2)-ethylGua lesion by the DNA polymerase eta is about 370-fold more efficient than by the DNA polymerase alpha, and extension from the N(2)-ethylGua:Cyt 3'-terminus by the DNA polymerase eta is about 3-fold more efficient than by the DNA polymerase alpha. Furthermore, the DNA polymerase eta preferably inserts the correct nucleotide Cyt opposite the N(2)-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 O(6)-ethylGua adduct by the DNA polymerases alpha and eta. In reactions containing both DNA polymerases alpha and eta, synthesis past the N(2)-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N(2)-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase alpha and lesion bypass catalyzed by DNA polymerase eta.