Supported by NCI/NIH 2R01CA098296, 2R56CA098296

The primary causes of sporadic human cancers are environmental. Aromatic amines are among the most notorious environmental chemicals that are implicated in the etiology of human cancers. Formation of arylamine-DNA adducts has been confirmed in various human tissues and is believed to induce chemical carcinogenesis. Hence it is imperative to elucidate how these lesions are repaired and replicated in vivo at the atomic and molecular levels. The key molecular players (adduct structures, polymerases, repair proteins) producing adverse outcomes must be identified, characterized, and understood in order to devise appropriate prevention and risk assessment strategies.

The long term goal of our research program is to elucidate the molecular mechanisms of DNA adduct-induced mutagenesis. Our working hypothesis is that adduct-induced conformational heterogeneity plays a critical role in determination of repair efficiency and fidelity of replication, and the detailed account of these balancing processes must be delineated. Aminofluorene (AF, Figure 1a) is a prototype arylamine-DNA adduct. We showed previously that AF exists in three sequence dependent conformational motifs (stacked [S], external B-type [B], or wedge [W])(Figure 1b; see also animation below), and that the population ratios of these conformations are related to mutational and repair outcomes in Escherichia coli. In addition, we established the ¹⁹F NMR/CD procedure as a powerful structural biology tool that is useful in dealing with the complex equilibriums that are otherwise difficult to study.

Figure 1. (a) Chemical structures of aminofluorene adduct AF, N- (2’-deoxyguanosin-8-yl) -2-aminofluorene and FAF, N- (2’-deoxyguanosin-8-yl) -7-fluoro-2-aminofluorene. (b) The major groove views for AF-induced B (B-type), S (stacked), and W (wedge) conformers. The modified dG and the complementary (dC for B and S, and dA for W) are indicated with red and green lines, respectively, and the carcinogenic aminofluorene is highlighted with grey CPK.

Our research programs focus on conformation-specific repair in the human nucleotide excision repair (NER) system, long-range sequence effects, thermodynamic probing of slipped mutagenic intermediates, and replication fork conformational heterogeneity. We employ not only well established ¹⁹F NMR/CD, EMSA and fluorescence spectroscopy procedures, but also innovative new methods such as isothermal calorimetry (ITC), differential scanning calorimetry (DSC), and surface plasmon resonance (SPR). These biophysical methodologies will be combined to help elucidate the protein-DNA interactions involved in NER and trans-lesion synthesis and understand the molecular details of cancer initiation.

(A) Conformation-specific NER in the human system. NER is a major pathway for the removal of potentially mutagenic lesions from the genome. We have formulated the concept that NER processing arylamine adducts could be conformation-specific: i.e., the S-conformation is more repair–prone than the B-conformation. This trend, however, was based on experiments conducted with conformer mixtures, not on pure conformers. In order to test this hypothesis, we examine a specific conformer (S, B, or W), rather than a mixture of conformers. We search for specific conformer motifs from a group of lesions of varying size, co-planarity, and sequence contexts, and assay them for their incision efficiencies in the human NER system. We use dynamic 19F NMR spectroscopy coupled with fluorinated FAF-modified DNA (Figure 1a).

(B) Conformation-specific protein-DNA interactions in the initial DNA damage recognition step. The molecular mechanisms by which carcinogen DNA adducts are recognized by the NER machinery are poorly understood. While there are some discrepancies in the reported affinity and specificities, compelling recent data support the suggestion that XPC-HR23B and XPA/RPA are both important NER proteins involved in the rate-limiting DNA damage recognition steps. We hypothesize that the binding affinities of the two protein complexes or at least one of them (most likely XPC-HR23B) to arylamine adducts are “conformation-specific” and contribute differently to subsequent NER processes including helix opening. We determine the molecular affinity between each adduct conformation (S, B, W) and XPC-HR23B or XPA and RPA. The protein-DNA interaction results are related to the overall NER incision efficiency data obtained in (A) above. We also define the thermodynamic basis of the differential recognition due to the conformation specificity. Our working theory is that the DNA duplex is a major substrate for the NER and that the steric and electrostatic aspects of groove environments of damaged DNA are critical determinants for initial recognition. We employ gel mobility shift, fluorescence spectroscopy, and SPR to determine Kd and thermodynamic properties as a gauge for their affinities

Click on the image to view animation.

(C) Thermodynamic probing of frameshift mutagenesis via a slippage pathway. The NarI recognition sequence (5’-G1G2CG3CN-3’) is the most recognized hot spot for frameshift mutagenesis induced by arylamines in E. coli plasmid pBR322. Although the three guanines in the sequence are equally chemically reactive, only AAF at G3 induces an unusually high frequency of -2 deletion mutations. The loss of a GC dinucleotide (5’-G1G2CN-3’), which occurs spontaneously at an extremely low frequency (<10^–8), is increased up to 10⁷-fold when an AAF residue is bound to G3. Moreover, the vulnerability to these mutations depends upon the nature of the nucleotide N (C >> T). AF has a much lesser (<10 X) propensity to induce frameshifts at G3. It has been shown that bypass of AAF at G3 requires Pol II for -2 deletion and Pol V for error-free trans-lesion synthesis. Our working hypothesis is that the conformational stability of a slipped mutagenic intermediate (SMI) is a major determining factor in the efficacy of -2 deletion mutation in the E. coli NarI sequence. We use dynamic ¹⁹F NMR/CD and DSC to carry out a systematic investigation of the thermodynamic contributions in the formation of SMIs by structurally related arylamine DNA adducts. Template-primer sequences are designed to mimic a progression of primers in simulated trans-lesion synthesis. We dissect the basic enthalpy and entropy contributions of the template-primers with dC at the replication fork, a configuration that is relevant to SMI formation.

(D) Conformational heterogeneity at the replication fork. The adduct structure at the replication fork is relevant to mutation, as it determines the nature of the incoming dNTP. We hypothesize that “adduct heterogeneity at the replication fork is sequence dependent and influences the short-term trans-lesion synthesis and polymerase binding affinity, leading to certain mutational outcomes.” We utilize the well-established AF-induced S/B-equilibrium to examine its impact on the thermodynamics and conformational kinetics of trans-lesion synthesis and polymerase binding affinity (Kd). To test the hypothesis, we study the thermodynamics and kinetics of AF-induced S/B heterogeneity during translesion synthesis (Figure 2), primer-extension kinetic experiments, and molecular probing of the replication fork heterogeneity upon polymerase binding (Klenow Fragment-exo- and Dpo4) by EMSA, ITC, and SPR.

Figure 2. Trans-lesion synthesis (TLS) models of BF complexed with AF-modified template/primer DNA: (a) B-conformer AF at the n+3 position and (b) W-conformer AF at the n+3 position. The carcinogenic AF moiety in red CPK; template and primer sequences in green and magenta, respectively.