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

Abstract

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 N2-dG adduct, 10S (+)-trans-anti-[BP]-N2-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 non-slippage 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 non-slippage 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.

Structure and Thermodynamic Insights on Acetylaminofluorene-Modified Deletion DNA Duplexes as Models for Frameshift Mutagenesis

“…64 It should be noted that Xu and coworkers have obtained some interesting structural insights on how a Y-family DNA polymerase may accommodate a slippage structure, however in this case it involved the spacious prokaryotic polymerase Dpo4. 65, 66 …”

Repair-Resistant DNA Lesions

“…168 The TLS past B[ a ]PDE- N 2 –dG (G*) adducts in vitro (169,170) and in cellular environments 171,172 have been documented. The insertion of nucleotides opposite G* other than dCTP gives rise to point mutations, while slipped frameshift intermediates can stimulate the bypass of G* without the insertion of any nucleotide, which can yield DNA duplexes with single nucleotide deletions.…”

Mechanism and Function of the Outer Membrane Channel TolC in Multidrug Resistance and Physiology of Enterobacteria

“…In contrast, very little structural information is available for ABC and MF pumps and the current assumptions are based on what we know about their TolC-independent homologs. For the macrolide efflux ABC-type MacB and its close homologs, direct interaction with TolC is still a possibility because these transporters also possess large periplasmic domains with structural features reminiscent of those of AcrB (Xu et al, 2009). However, the type I secretion ABC transporters and MF transporters seem to be unable to reach the periplasmic tip of TolC.…”