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, 31 (11), 1260-1268

Lesion Sensing During Initial Binding by Yeast XPC/Rad4: Toward Predicting Resistance to Nucleotide Excision Repair

Affiliations

Lesion Sensing During Initial Binding by Yeast XPC/Rad4: Toward Predicting Resistance to Nucleotide Excision Repair

Hong Mu et al. Chem Res Toxicol.

Abstract

Nucleotide excision repair (NER) excises a variety of environmentally derived DNA lesions. However, NER efficiencies for structurally different DNA lesions can vary by orders of magnitude; yet the origin of this variance is poorly understood. Our goal is to develop computational strategies that predict and identify the most hazardous, repair-resistant lesions from the plethora of such adducts. In the present work, we are focusing on lesion recognition by the xeroderma pigmentosum C protein complex (XPC), the first and required step for the subsequent assembly of factors needed to produce successful NER. We have performed molecular dynamics simulations to characterize the initial binding of Rad4, the yeast orthologue of human XPC, to a library of 10 different lesion-containing DNA duplexes derived from environmental carcinogens. These vary in lesion chemical structures and conformations in duplex DNA and exhibit a wide range of relative NER efficiencies from repair resistant to highly susceptible. We have determined a promising set of structural descriptors that characterize initial binding of Rad4 to lesions that are resistant to NER. Key initial binding requirements for successful recognition are absent in the repair-resistant cases: There is little or no duplex unwinding, very limited interaction between the β-hairpin domain 2 of Rad4 and the minor groove of the lesion-containing duplex, and no conformational capture of a base on the lesion partner strand. By contrast, these key binding features are present to different degrees in NER susceptible lesions and correlate to their relative NER efficiencies. Furthermore, we have gained molecular understanding of Rad4 initial binding as determined by the lesion structures in duplex DNA and how the initial binding relates to the repair efficiencies. The development of a computational strategy for identifying NER-resistant lesions is grounded in this molecular understanding of the lesion recognition mechanism.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Crystal structure of the yeast orthologue of human XPC productively bound to CPD containing-DNA with mismatched thymines (PDB ID: 2QSG). The crystal structure is shown in cartoon representation. The TGD is yellow, the R4BD (Rad4/XPC binding domain in Rad23) is beige, BHD1 is marine, BHD2 is orange, BHD3 is dark green, and the DNA is light gray. The unresolved CPD (red) and BHD2 (orange) hairpin tip are indicated by dashed lines. The mismatched thymines (blue) that are flipped into their binding pockets are also shown in a zoomed-in view showing the surface of the binding pockets.
Figure 2
Figure 2
Lesion structures. (A) Chemical structures of the selected lesions: 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG), 10S (+)-trans-anti-B[a]P-N2-dG [(+)-trans-B[a]P-dG], 10R (−)-trans-anti-B[a]P-N2-dG [(−)-trans-B[a]P-dG], 14R-(+)-trans-anti-dibenzo[a,l]pyrene-N2-dG (14R-DB[a,l]P-dG), 14R-(+)-trans-anti-dibenzo[a,l]pyrene-N6-dA (14R-DB[a,l]P-dA), and N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG). The benzylic ring is denoted by “A”. The * designates that the base is modified. (B) NMR/MD-derived structures of lesion-containing duplexes. The structures of the central 5-mers are shown in cartoon and sticks, with hydrogen atoms and backbone phosphate oxygens hidden. The lesion-containing base and its partner are blue, and the adduct is red. The view is into the minor groove. Full details for the NMR/MD-derived structures are reviewed in ref (32) and given in refs (, , , and −39). The structural properties are fully summarized in Supporting Information. For the cis-B[a]P-dG cases, key differences entail the partner base identity and position or its absence. For the minor groove positioned trans-B[a]P-dG, the key difference is the 5′ vs 3′ orientation of the B[a]P ring system. For the PhIP-C8-dG, the mobile phenyl ring protruding in the minor groove is a key structural feature. For the bulky 14R-DB[a,l]P adducts, key differences are the intercalation from the minor groove with ruptured G*:C base pair for the dG* adduct but intercalation from the major groove with Watson–Crick pairing maintained for the dA* adduct.
Figure 3
Figure 3
Structures and initial binding descriptors obtained from MD simulations and experimental NER excision efficiencies for the lesion-containing duplexes. (A) The AS volumes occupied by BHD2 in the lesion-containing DNA are shown in orange bars; this volume reflects the curvature and surface area of the DNA minor groove bound by BHD2. The means and standard deviations for the block average values of untwist angles (detailed in Figure S1 and Supporting Information Methods) are shown in cyan bars and dark red lines. The relative NER excision efficiencies are in pink bars with the cis-B[a]P-dG:dC duplex assigned a relative value of 100. NER data are reviewed in ref (32) and are given in refs ( and −31). (B–D) Best representative structures of the initial binding states from the MD trajectories. The structures are shown in cartoon with F556, F597, and F599 side chains in spheres. The base pairs used for calculation of untwist angles are in cyan. Insets depict zoomed-in views of captured partner strand base for the well-repaired cases, with BHD2 and BHD3 in surface representation. Movies S1S10 show these initial binding state structures.

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