Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

Abstract

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo [a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N 2 -dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3±0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5′-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/ bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N 2 -dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/ stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.

Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

“…In NER experiments using prokaryotic UvrA Bca B Bca C Tma proteins, the ratio was reported to be 2.3 ± 0.3 at 37 °C and 1.7 ± 0.4 at 55 °C. This loss of discrimination at the higher temperature is believed be due to the increased dynamic conformational flexibility at the lesion site at 55 °C; this has a greater impact in the more rigid CG 6 C sequence context of duplex III than on the more flexible TG 6 T sequence context of duplex IV [59]. In the case of E. coli UvrA E.coli UvrB E.coli UvrC E.coli , however, the ratio at 37 °C was smaller (1.7 ± 0.1) in E. coli UvrABC incision experiments [9].…”

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene

“…Several studies have been conducted on the effects of nearest-neighbor and distantneighbor sequences on the repair rate of BPDE-dG. The data have been interpreted within the context of structural deformity and duplex backbone flexibility caused by the adduct in various sequence contexts, and some general rules have been derived (24)(25)(26)(27). Although these pioneering studies have been quite useful, some of the rules were derived from the excision rates of the BPDE-dG adduct from in vitro experiments with human cellfree extracts and oligonucleotide duplexes 135 bp in length (28).…”

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex

“…There are a few in vitro studies that also suggest that cleavage is not completely sequence-independent; for example, cleavage of an identical sequence where an acetylaminofluorene adduct positioned at three distinct guanines led to significant variation in incision efficiency (52). Similar differences were found for benzo[a]pyrenyl-guanine lesions placed at various positions (53,54). In addition de Laat et al (32) presented evidence for sequence specificity by showing that splayed arm substrates with distinct sequence composition around the junction were cleaved at distinct positions in the stem sequence, arguing that the incision position is somewhat dictated by the DNA sequence.…”

“…This well orchestrated cleavage process (61-63) results in the removal of 24 -32 nucleotides both in vitro and in vivo. The substantial variation in both cleavage position with respect to the damage and the length of the removed sequence (5, 64) suggests some heterogeneity in the cleavage mechanism, which is underscored by flanking sequence-dependent differences in cleavage efficiency in vitro (43,(52)(53)(54)(55) and in vivo (56).…”