Visualizing sequence-governed nucleotide selectivities and mutagenic consequences through a replicative cycle: processing of a bulky carcinogen N2-dG lesion in a Y-family DNA polymerase.

Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N(2)-dG adduct, 10S-(+)-trans-anti-[BP]-N(2)-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5'-C[BP]G*G-3' sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5'-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5'-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

Nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG adduct in a Y-family DNA polymerase: a 5'-slippage mechanism.

The Y-family DNA polymerase Dpo4, from the archaeon bacterium Sulfolobus solfataricus, is a member of the DinB family, which also contains human Pol kappa. It has a spacious active site that can accommodate two templating bases simultaneously, with one of them skipped by the incoming dNTP. Assays of single dNTP insertion opposite a benzo[ a]pyrene-derived N (2)-dG adduct, 10 S(+)- trans- anti-[BP]- N (2)-dG ([BP]G*), reveal that an incoming dATP is significantly preferred over the other three dNTPs in the TG 1*G 2 sequence context. Molecular modeling and dynamics simulations were carried out to interpret this experimental observation on a molecular level. Modeling studies suggest that the significant preference for dATP insertion observed experimentally can result from two possible dATP incorporation modes. The dATP can be inserted opposite the T on the 5' side of the adduct G 1*, using an unusual 5'-slippage pattern, in which the unadducted G 2, rather than G 1*, is skipped, to produce a -1 deletion. In addition, the dATP can be misincorporated opposite the adduct. The 5'-slippage pattern may be generally facilitated in cases where the base 3' to the lesion is the same as the adducted base.

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase.

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.