Variable impact of conformationally distinct DNA lesions on nucleosome structure and dynamics: Implications for nucleotide excision repair

Abstract

The packaging of DNA in nucleosomes presents a barrier for biological transactions including replication, transcription and repair. However, despite years of research, how the DNA is freed from the histone proteins and thereby allows the molecular machines to access the DNA remains poorly understood. We are interested in global genomic nucleotide excision repair (GG-NER). It is established that the histones are obstacles to this process, and DNA lesions are repaired less efficiently in nucleosomes than in free DNA. In the present study, we utilized molecular dynamics simulations to elucidate the nature of the distortions and dynamics imposed in the nucleosome by a set of three structually different lesions that vary in GG-NER efficiencies in free DNA, and in nucleosomes [Shafirovich, Geacintov, et. al, 2019]. Two of these are bulky lesions derived from metabolic activation of the environmental carcinogen benzo[a]pyrene, the 10R (+)-cis-anti-B[a]P-N²-dG and the stereoisomeric 10S (+)-trans-anti-B[a]P-N²-dG, which respectively adopt base-displaced/intercalated and minor groove-aligned conformations in DNA. The third is a non-bulky lesion, the 5'R-8-cyclo-2'-deoxyguanosine cross-link, produced by reactive oxygen and nitrogen species; cyclopurine lesions are highly mutagenic. These adducts are placed near the dyad axis, and rotationally with the lesion-containing strand facing towards or away from the histones. While each lesion has distinct conformational characteristics that are retained in the nucleosome, a spectrum of structural and dynamic disturbances, from slight to substantial, are displayed that depend on the lesion's structure and position in the nucleosome. We hypothesize that these intrinsic structural and dynamic distinctions provide different signals to initiate the cascade of chromatin-opening processes, including acetylation and other post translational modifications, remodeling by ATP-dependent complexes and spontaneous unwrapping that regulate the rate of access to the lesion; this may translate ultimately into varying GG-NER efficiencies, including repair resistance when signals for access are too weak.

Keywords: 5’R-8-cyclo-2’-deoxyguanosine cross-link; Benzo[a]pyrene diol epoxide-derived DNA adducts; DNA damage; Molecular dynamics; Nucleosome; Nucleotide excision repair; Rotational and translational setting.

Figure 1.. Structure of unmodified nucleosome, and the chemical and solution structures of the lesions.

(A) Unmodified nucleosome modeled based on PDB [58] ID 1KX5 [59], see Methods. Lesion positions are designated as red stars. The IN position has the lesion two base pair steps from the dyad axis (at SHL = −0.25); the lesion-containing DNA strand facing histones and its partner faces the solvent. In the OUT position, the lesion is seven base pair steps from the dyad axis (at SHL = − 0.75); the lesion-containing DNA strand faces the solvent while its partner faces histones. Note that the lesion-containing DNA base pair confronts a different histone landscape in the IN and OUT positions. The left panel shows the view down the superhelix axis while the right panel shows a rotated view that reveals the two gyres with histones rendered in surface. Histones H3 and H4 are from Chains A and B respectively in PDB ID: 1KX5. A brief review of nucleosome structure is given by Cutter and Hayes [70]. (B) Chemical structures of lesions investigated. The benzylic ring of the B[a]P ring system is designated as “A”, and “dR” is deoxyribose. (C) Conformations of the lesion-containing DNAs [49, 53, 57]. Views are looking into the DNA minor groove. Color codes: DNA, grey, except damaged dG or its corresponding unmodified nucleoside, salmon, and its partner dC, marine; histone H3, yellow and H4, teal. Hydrogen atoms are omitted for clarity.

Figure 2.. Best representative structures of lesion-containing nucleosomes and dynamics of partner dC

Base-displaced/Intercalated cis-B[a]P-dG with Watson-Crick pairing ruptured (A and B); (A): IN: the partner dC faces solvent, while the damaged dG in the minor groove faces the histones, forming two new hydrogen bonds with nearby Arg 45 of histone H4. (B) OUT: the confined partner dC faces histones with its backbone phosphate oxygens rotated toward the minor groove, and OP2, that is closer to H4 Arg 45, forms a new hydrogen bond with it. The adducted dG, now faces the solvent, and is inherently restrained due to its linkage to the benzo[a]pyrenyl ring system that is intercalated between adjacent base pairs. Minor groove-aligned trans-B[a]P-dG with Watson-Crick pairing maintained and minor groove enlarged (C and D); (C): IN: the histone-facing B[a]P rings that enlarge the minor groove cause shifting of the partner strand, which forms one partial and one full new hydrogen bond with Arg 35 and Arg 39 of the H4 histone. (D): OUT: the B[a]P rings face the solvent and there is no change in hydrogen bond contacts with histones. In both IN and OUT cases, the B[a]P rings are shielded on one face by the minor groove wall. Small and constrained cross-link R-cdG with Watson-Crick pairing maintained (E and F) (E): IN: hydrogen bond interactions are unchanged (F):OUT: two hydrogen bonds are lost between the DNA backbone of the partner strand and the nearby Arg 35 of histone H4, while one is gained between Arg 39 and this partner backbone, yielding a net loss of one hydrogen bond; this is caused by the shifting of the partner dC as it strives to maintain Watson-Crick pairing with R-cdG. Color codes are the same as in Figure 1. Movies S1 and S2 reveal the best representative structures of the cis-B[a]P-dG-OUT and -IN, respectively.

Figure 3.. Partner dC flipped into the major groove is more dynamic for cis-B[a]P-dG-IN.

(Left Panels) Time dependence of flipping pseudo-dihedral angles of partner dC for (A) IN-facing lesion, and (B) OUT-facing lesion. Mean values and standard deviations are given. The much greater dynamics is revealed in the larger range and standard deviation for the IN-facing lesion where partner dC faces solvent. With this greater range, the dC samples a larger span in the major groove. This is shown in the right panels with superimposed positions of the dCs at pseudo-dihedral angles of the mean values (blue), and plus/minus their standard deviations (purple and green, respectively). These structures were superimposed by aligning all DNA atoms in the duplex trimer centered at the lesion-containing base pair. In the unmodified cases (left panels, black; structures not shown), the partner dC is stacked in; the flipping pseudo-dihedral angles, ~ 35 °, differ slightly for the IN vs. OUT cases, because they have different SHL positions and local sequence contexts. The definition of the flipping pseudo-dihedral angle is shown in Figure S2, in Supporting Information. The larger the flipping pseudo-dihedral angle, the greater the extrusion into the major groove. Color codes are the same as in Figure 2.