Fig 1.
Adduct structures and sequences.
(A) Structure of AAF [N-(2’-deoxyguanosin-8-yl)-2-acetylaminofluorene], AF [N-(2’-deoxyguanosin-8-yl)-2-aminofluorene] and fluoro models, FAAF [N-(2-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene], FAF [N-(2’-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]; (B) Fully-paired 16-mer duplexes containing the central NarI sequence (CGGCGCC) used in SPR, EMSA and in vitro NER constructs illustrating the placement of the adducted bases at G1, G2, and G3 positions; (C) Major groove views of the B-, S-, and W-conformers of AAF. Modified-dG (red), dC (green) opposite the lesion site (orphaned C), fluorene (grey CPK), N-acetyl (magenta).
Table 1.
Correlation of XPC-RAD23B binding and dissociation parameters, melting temperature, and hNER efficiencies of FAAF-modified NarI substrates.
Table 2.
Binding and dissociation parameters of UvrA2 binding to FAAF-modified NarI substrates.
Fig 2.
NER dual incision at adducts in the NarI sequence in the human NER system.
Plasmids containing site-specific mono-FAAF (lanes G1, G2, G3) or di-FAAF adducts (lanes G1G2, G2G3, G1G3), were incubated with HeLa whole-cell extracts. Detection of excision products was monitored by 3’-end-labeling using a complementary oligonucleotide containing a 5’-GGGG base overhang. The reaction products were resolved on a 12% denaturing polyacrylamide gel run under constant current. The range of excision products is indicated on the left of the gel.
Fig 3.
The efficiencies of NER at adducts in the NarI sequence in the human NER system.
The relative incision rates of mono-FAAF and di-FAAF adducts in the histogram were calculated by normalizing the mono- (yellow) and di-adducts (blue) relative to the NarI-G2G3 FAAF value for the NarI-G1 FAAF value for the human system. Quantification of NER efficiencies was from at least three independent experiments.
Fig 4.
XPC binding to damaged DNA in the NarI sequence.
(A) Representative image of XPC binding to NarI-G1 in an EMSA assay. XPC protein, at increasing concentration, was incubated with a FAAF-damaged 55-bp oligo. Sensograms showing XPC binding kinetics to mono-FAAF (B) and to di-FAAF adducted substrates (C). SPR responses were recorded for the binding of XPC NER protein (5, 2.5, 1.25, 0.625, 0.313, and 0.156 nM) to FAAF-modified full DNA duplexes. The recorded data are displayed as black lines while red lines represent curve fitting. The half-life (t1/2) is indicated above the curves (in yellow box) and is defined as the time it takes for half of the XPC-DNA complex to dissociate. The fitted curves obtained from fittings using a one-independent site model (“Scrubber”) are displayed (See Methods).
Fig 5.
Comparison of hNER efficiency and half-life (t1/2) of the XPC-DNA complex.
The data points were analyzed independently as mono- or di-FAAF adduct groups and the two dashed lines indicates the group trends. Mono- and di-adducts are indicated on the right.
Fig 6.
Proposed model for XPC interaction with DNA-adduct site.
(A) 3D-printed model (not simulated) to illustrate the potential binding of the yeast XPC-RAD23B ortholog, Rad4/Rad23, to the mono-G1-FAAF duplex based on PDB ID 2QSG. The β-hairpin domains (BHD2 and BHD3) and the transglutaminase-homology domain (TGD), which are involved in protein-DNA interaction, are indicated. The domains were adapted from previous crystal structure analysis by Min et al. [24]. The duplex sequence used in this model is identical with that of Min’s crystal work except that the CPD lesion was replaced by FAAF-G1 (yellow *, as shown). The site of additional FAAF in the di-G2G3-adduct is designated in red asterisk. The insertion of the BHD3 β-hairpin was accompanied with flipping of the mismatched bases (cyan) on the complimentary sequence. (B) A schematic illustrating the proposed mechanism of action where XPC is loosely bound to mono-FAAF adducted DNA (left) or tightly bound to di-FAAF adducted DNA (right). Following dissociation of XPC from the damage site subsequent NER factors are recruited to complete the excision of the damaged base; however, in the di-adduct situation XPC is retained on the damaged DNA, delaying successful NER completion.