Nucleotide excision repair of 2-acetylaminofluorene- and 2-aminofluorene-(C8)-guanine adducts: molecular dynamics simulations elucidate how lesion structure and base sequence context impact repair efficiencies

Abstract XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

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Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7069 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7069-7076 ◽

Cited By ~ 99

Author(s):

M T Hess ◽

D Gunz ◽

N Luneva ◽

N E Geacintov ◽

H Naegeli

Keyword(s):

Nucleotide Excision Repair ◽

Dna Adducts ◽

Excision Repair ◽

Dna Conformation ◽

Repair Processes ◽

Repair Enzymes ◽

Diol Epoxide ◽

Nucleotide Excision ◽

Guanine Adducts ◽

Repair Activity

Human nucleotide excision repair processes carcinogen-DNA adducts at highly variable rates, even at adjacent sites along individual genes. Here, we identify conformational determinants of fast or slow repair by testing excision of N2-guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G x C-->T x A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (+)-trans-BPDE-N2-dG adduct 15 times less efficiently than a standard acetylaminofluorene-C8-dG lesion in the same sequence. No difference was observed between (+)-trans- and (-)-trans-BPDE-N2-dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (+)-cis- or (-)-cis-BPDE-N2-dG. Conversely, excision of (+)-cis- and (-)-cis- but not (+)-trans-BPDE-N2-dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N2-dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.

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