Differential Nucleotide Excision Repair Susceptibility of Bulky DNA Adducts in Different Sequence Contexts: Hierarchies of Recognition Signals

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Abstract

The structural origin underlying differential nucleotide excision repair (NER) susceptibilities of bulky DNA lesions remains a challenging problem. We investigated the 10S (+)-trans-anti-[BP]-N2-2′-deoxyguanosine (G⁎) adduct in double-stranded DNA. This adduct arises from the reaction, in vitro and in vivo, of a major genotoxic metabolite of benzo[a]pyrene (BP), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, with the exocyclic amino group of guanine. Removal of this lesion by the NER apparatus in cell-free extracts has been found to depend on the base sequence context in which the lesion is embedded, providing an excellent opportunity for elucidating the properties of the damaged DNA duplexes that favor NER. While the BP ring system is in the B-DNA minor groove, 5′ directed along the modified strand, there are orientational distinctions that are sequence dependent and are governed by flanking amino groups [Nucleic Acids Res. 35 (2007), 1555–1568]. To elucidate sequence-governed NER susceptibility, we conducted molecular dynamics simulations for the 5′-…CG⁎GC…, 5′-…CGG⁎C…, and 5′-…TCG⁎CT… adduct-containing duplexes. We also investigated the 5′-…CG⁎IC… and 5′-…CIG⁎C… sequences, which contain “I” (2′-deoxyinosine), with hydrogen replacing the amino group in 2′-deoxyguanosine, to further characterize the structural and dynamic roles of the flanking amino groups in the damaged duplexes. Our results pinpoint explicit roles for the amino groups in tandem GG sequences on the efficiency of NER and suggest a hierarchy of destabilizing structural features that differentially facilitate NER of the BP lesion in the sequence contexts investigated. Furthermore, combinations of several locally destabilizing features in the hierarchy, consistent with a multipartite model, may provide a relatively strong recognition signal.

Introduction

The factors that invoke nucleotide excision repair (NER) of bulky DNA adducts have been the object of intense interest for a number of years.1, 2, 3, 4, 5, 6, 7, 8, 9 Various experimental studies have suggested the importance of distortions, such as kinks in the damaged DNA,10 impaired Watson–Crick hydrogen-bonding,4 flipped-out nucleotides on the partner strand,9 enhanced local dynamics,11, 12, 13, 14 and thermodynamic destabilization,5, 15 including a multipartite model involving combinations of various local thermodynamically destabilizing DNA distortions.14, 16 Bulky lesions derived from the binding of benzo[a]pyrene (BP) metabolites to DNA that do not cause thermal destabilization17 have been found to be resistant to repair by human NER in cell-free extract assays.15, 16, 18

A set of DNA duplexes containing the same BP-derived lesion but in different sequence contexts and with differing susceptibilities to human NER provides a fascinating opportunity to explore the structural features that produce observed differences in NER. These duplexes all contain the highly mutagenic major 10S (+)-trans-anti-[BP]-N2-2′-deoxyguanosine (dG) adduct (Fig. 1a) produced from the reaction of the strongly tumorigenic metabolite of BP, (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BP diol epoxide],19 with the exocyclic amino group of guanine in DNA.20, 21, 22 These duplexes differ in sequence contexts surrounding the lesion: 5′-…GCG⁎GC… (G6⁎G7), 5′-…CGG⁎CC… (G6G7⁎), 5′-...TCG⁎CT… (CG⁎C-I), 5′-…ACG⁎CA… (CG⁎C-II), 5′-…ATG⁎TA… (TG⁎T), and 5′-…CIG⁎CC… (I6G7⁎), with the asterisk designating the modified bases and “I” representing 2′-deoxyinosine (Fig. 1b). Relative repair susceptibilities in a eukaryotic NER assay are in the ratio of 4.1 ± 0.3 (G6⁎G7):2.4 ± 0.2 (TG⁎T):1.7 ± 0.2 (G6G7⁎):1.5 ± 0.2 (CG⁎C-II):1.3 ± 0.2 (I6G7⁎):1.0 (CG⁎C-I).23 The GG tandem guanine sequence has been the object of unusual interest because it is a mutational hotspot in mouse embryonic NIH 3T3 cells,24 mammalian V-79 cells,25 and the SupF gene of an Escherichia coli plasmid.26, 27

Structural studies by high-resolution NMR methods have been carried out for the G6⁎G7, G6G7⁎, and CG⁎C-I duplexes.28, 29 These revealed that the BP ring system in all cases is located in the minor groove, oriented toward the 5′ direction of the modified strand. However, subtle structural differences are observed in the three sequence contexts because of sequence-dependent steric competition between the bulky amino groups of the guanines flanking the modified G⁎ and the BP residue, which is positioned in the minor groove in all cases.28, 29 Additionally, molecular dynamics (MD) studies for the CG⁎C-II and TG⁎T sequence contexts (Fig. 1b) indicated that the BP ring system also resides in the minor groove, 5′ directed along the modified strand but with more dynamic flexibility in the TG⁎T case.14, 30

Our goal in the present study was to determine how the 10S (+)-trans-anti-[BP]-N2-dG adduct impacts the dynamic structural properties of these damaged duplexes in a sequence-dependent manner; we aimed to gain deeper understanding of the properties that favor NER by comparing our findings with the experimental NER results. We employed 10.0 ns of unrestrained MD simulations to provide ensembles of structures for the G6⁎G7, G6G7⁎, and CG⁎C-I duplexes; these ensembles, based on NMR solution structures, yielded new insights into the local dynamic properties of the duplexes. Our results delineated the roles of the exocyclic amino groups of guanines flanking the lesion on the duplex dynamic properties. We further substantiated these findings by replacing the flanking G's with I's (2′-deoxyinosine, with hydrogen replacing the amino group in dG) for the GG sequence contexts. Coupled with previous MD results for the CG⁎C-II and TG⁎T sequence contexts,14 our investigations suggest a hierarchy of destabilizing structural features that thermodynamically facilitate the recognition of the BP-modified duplexes by the NER apparatus for the sequences we considered. Dynamic, episodically denatured Watson–Crick hydrogen bonding at the base pair on the 5′ side of the lesion provides the strongest signal, followed by dynamic local untwisting accompanied by a large Roll/bend. Enlarged minor groove widths and modest perturbations of hydrogen bonding at and adjacent to the lesion site are common to all the damaged duplexes, with only slight sequence-dependent dynamic differences, and are weaker recognition factors. Furthermore, various elements from the hierarchy can be combined in a multipartite model to produce a relatively strong NER recognition signal. We propose that the varied destabilizing features differentially facilitate the insertion of a β-hairpin from the human recognition factor XPC (the homolog of yeast Rad431) between the two strands of the BP-modified duplexes at or near the lesion site.

Section snippets

Results

In this study, we utilized the AMBER 8.0 simulation package32 to produce 10.0 ns of MD ensembles for the 10S (+)-trans-anti-[BP]-N2-dG adduct in the G6⁎G7, G6G7⁎, and CG⁎C-I duplexes, as well as their unmodified control duplexes G6G7 and CGC-I. We used the high-resolution NMR solution structures28, 29 as initial models and generated unrestrained MD ensembles that preserved the NMR structural features for each lesion-containing duplex yet provided fuller views of their dynamic nature. We also

Differences in steric hindrance from exocyclic amino groups of guanines flanking the lesion on either side explain distinct sequence-dependent structural characteristics and dynamic phenomena

In the G6⁎G7 duplex, the amino group of the 3′-flanking G7 leads to episodic unpairing of the 5′-flanking C5:G20 base pair, while in the G6G7⁎ duplex, the amino group of the 5′-flanking G6 causes unusual flexible local untwisting concomitant with an increased local Roll/bend. The importance of the guanine amino group is substantiated by studies in which 2′-deoxyinosine replaces the dG adjacent to the lesion; this relieves the steric hindrance in the G6⁎G7 and G6G7⁎ duplexes by replacing the

Conclusions

We employed MD simulations to uncover dynamic structural correlations with repair susceptibilities for the 10S (+)-trans-anti-[BP]-N2-dG adduct in several sequence contexts. By utilizing the identical minor groove-aligned lesion in a number of sequence contexts that exhibit varying susceptibilities to removal by the human NER system in cell-free extracts, we propose hierarchies of structural signals for invoking NER with differential efficiencies in these sequence contexts. We found that

Starting structures

The starting models were the minor groove conformations of the NMR solution structures for the 10S (+)-trans-anti-[BP]-N2-dG adduct in the G6⁎G7, G6G7⁎,28 and CG⁎C sequence contexts.29 The starting models for the unmodified DNA duplexes were built using standard B-DNA in INSIGHTII 2005 (Accelrys, Inc.) and were subjected to energy minimization in SANDER in the AMBER 8.0 simulation package.32 These initial models are shown in Fig. S11.

Force field

MD simulations were carried out employing SANDER in the AMBER

Acknowledgements

This work was supported by the National Cancer Institute, National Institutes of Health, through grants CA28038 (S.B.), CA099194 (N.E.G.), and CA046533 (D.J.P). Partial support for computational infrastructure and systems management was also provided by grant CA75449 (S.B.). The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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