We recently proposed a mechanism for why dAMP is primarily inserted opposite both T's of photoproducts of TT sites by T7 DNA polymerase [Smith, C. A., Baeten, J., and Taylor, J.-S. (1998) J. Biol. Chem., 273, 21933-21940] that was based on analysis of a recent crystal structure of a complex of this enzyme with a template, a primer, and a dideoxynucleotide. We proposed that indiscriminate insertion of dAMP opposite the 3'-T of each photoproducts takes place via a transient abasic site-like intermediate, with the photoproduct outside the active site, whereas insertion of dAMP opposite the 5'-T takes place with the photoproduct inside the active site. To obtain further support for this mechanism, we have investigated the selectivity of dNMP and pyrene nucleotide (dPMP) insertion opposite each T of the cis,syn, trans,syn-I, trans,syn-II, (6-4), and Dewar photoproducts of TT and opposite a tetrahydrofuran abasic site analogue by the exonuclease-deficient T7 DNA polymerase, Sequenase Version 2.0. Selectivity was determined by a direct competition assay that makes use of a stacked gel to resolve the various extension products. Pyrene nucleotide was chosen for investigation because it has been previously shown to be selectively inserted opposite abasic sites and was therefore expected to probe whether the photoproducts were inside the active site during a particular insertion step. In accord with the proposed mechanism, dPMP was inserted in preference to dAMP opposite the 3'-T of all the photoproducts with the exception of the trans,syn-I product, whereas dAMP was inserted in preference to dPMP opposite the 5'-T of all the photoproducts. In addition to supporting the proposed mechanism, these results suggest that pyrene nucleotide may be a useful probe for investigating the mechanism of DNA damage bypass by polymerases and for characterizing their active sites.