Free Energy Profiles of Base Flipping in Intercalative Polycyclic Aromatic Hydrocarbon-Damaged DNA Duplexes: Energetic and Structural Relationships to Nucleotide Excision Repair Susceptibility

Abstract

The crystal structure of Rad4/Rad23, the yeast homolog of the human nucleotide excision repair (NER) lesion recognition factor XPC-RAD23B (Min and Pavletich, (2007) Nature 449:570–575) reveals that the lesion-partner base is flipped out of the helix and binds the protein. This suggests the hypothesis that flipping of this partner base must overcome a free energy barrier, which constitutes one element contributing to changes in the thermodynamic properties induced by the DNA damage and sensed by the recognition protein. We explored this hypothesis by computing complete flipping free energy profiles for two lesions derived from the procarcinogenic polycyclic aromatic hydrocarbons (PAHs), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), R-trans-anti-DB[a,l]P-N6-dA (R-DB[a,l]P-dA) and R-trans-anti-B[a]P-N6-dA (R-B[a]P-dA), and the corresponding unmodified duplex. The DB[a,l]P and B[a]P adducts differ in number and organization of their aromatic rings. We integrate these results with prior profiles for the R-trans-anti-DB[a,l]P-dG adduct (Zheng et al., (2010) Chem. Res. Toxicol. 23:1868–1870). All adopt conformational themes involving intercalation of the PAH aromatic ring system into the DNA duplex; however, R-DB[a,l]P-dA and R-B[a]P-dA intercalate from the major groove, while R-DB[a,l]P-dG intercalates from the minor groove. These structural differences produce different computed van der Waals stacking interaction energies between the flipping partner base with the lesion aromatic ring system and adjacent bases; we find that the better the stacking, the higher the relative flipping free energy barrier and hence lower flipping probability. The better relative NER susceptibilities correlate with greater ease of flipping in these three differently intercalated lesions. In addition to partner base-flipping, the Rad4/Rad23 crystal structure shows that a protein-β-hairpin, BHD3, intrudes from the major groove side between the DNA strands at the lesion site. We present a molecular modeling study for the R-DB[a,l]P-dG lesion in Rad4/Rad23 showing BHD3 β-hairpin intrusion with lesion eviction, and we hypothesize that lesion steric effects play a role in the recognition of intercalated adducts.

The relationships between XPC binding to conformationally diverse DNA adducts and their excision by the human NER system: Is there a correlation?

“…In Figure 9A we have utilized the NMR solution structure of the well-repaired[21] R-trans -DB[ a,l ]P-G lesion [35] that is intercalated into the DNA duplex from the minor groove side, and the structure of the apo Rad4-Rad23 crystal (PDB ID: 2QSF, [18]) to illustrate the pre-bound states of the damaged duplex and the Rad4-Rad23. In Figure 9B, we have modeled (details given in Cai et al 11]) an extruded structure for the R-trans -DB[ a,l ]P-G lesion based on the thymine dimer- containing crystal structure of Rad4-Rad23 (PDB ID: 2QSG, [18]). We envision that the BHD3 β-hairpin intrusion through the major groove expels the minor grove intercalated lesion via the minor groove route (Figure 9).…”

“…We envision that the BHD3 β-hairpin intrusion through the major groove expels the minor grove intercalated lesion via the minor groove route (Figure 9). We have speculated that the repair-resistant DB[ a,l ]P-A adducts that are intercalated into the DNA duplex from the major groove [36] may sterically impede BHD3 intrusion [11]. Future studies will provide essential insights on how productive recognition of bulky PAH-derived lesions by XPC-RAD23B occurs and how XPC-RAD23B interacts with such repair-resistant lesions.…”

“…(B) the BHD3 hairpin loop has inserted into the duplex from the major groove side and extruded the R-trans -DB[ a,l ]P-G stacked with its 3’ adjacent dC (purple in A and B) through the minor groove. The Rad4/Rad23 complex containing a cis-syn thymine dimer (PDB ID: 2QSG, [18]) was remodeled to replace the thymine dimer (whose coordinates were missing) with the R-trans -DB[ a,l ]P-G-C dinucleotide from the NMR structure (PDB ID:2LZK, [35]) followed by 10 ns of molecular dynamics simulation [11] The missing BHD3 hairpin loop in (A) was modeled based on its structure in (B). This model suggests how productive binding may take place for a well-repaired PAH-derived NER substrate.…”

“…However, neither DDB2-DDB1 nor centrin 2 are required for the recognition of DNA adducts in cell extracts in vitro . The binding of the XPC-RAD23B heterodimer to the damaged DNA is the initial event [9, 10] that triggers the ensuing stepwise cascade of NER steps [11–15]. In order for the full processing of the DNA adducts to occur, it is believed that a two-step (bipartite) mechanism is required: (1) recognition by XPC-RAD23B and (2) the subsequent verification step that involves the helicase activity of XPD in TFIIH, the next multi-protein NER factor that binds to the XPC-RAD23B -DNA complex.…”

Repair-Resistant DNA Lesions

“…Since this intrusion of the BHD3 hairpin occurs from the major groove, the interaction of these bulky, major groove intercalated adenine adducts likely obstructs the hairpin insertion, which is also resisted by the stacking interactions of the DB[ a , l ]P aromatic ring system with adjacent base pairs. 196 Together, these phenomena could be sufficient to inhibit productive binding by XPC. The overall binding affinities of the DNA lesion-sensing XPC-RAD23B factor to the 14 R and 14 S trans -DB[ a , l ]PDE- N 6 –dA are experimentally indistinguishable.…”