Of particular interest is the fact that metabolic activation of PAH compounds in mammalian cells is highly stereoselective.  An example of the kind of diol epoxide stereoisomers we are studying is depicted in Figure 3. 

The two diol epoxides are mirror images of one another.  What is fascinating about these two enantiomers is that they form adducts of different structures when they react chemically with DNA (Figure 4).  We have found that DNA adducts of different structures (conformations) are excised by human and bacterial DNA repair enzymes at different rates, thus representing differences in their impact  on the regulatory mechanisms governing the replication of cells and their potential transformation into cancerous forms. Figure 4 depicts the most important structural motifs we discovered.  Most of these were originally discovered through our collaboration with Dinshaw Patel at Memorial Sloan Kettreing Cancer Center (see below group) where the structures shown were determined by NMR methods.  The major structural types can be divided into

(a) minor groove: the PAH is partially accessible to solvent,

(b) classical intercalation (“sandwich” structure), the PAH residue is protected from the solvent enviromnment, and

(c) base-displaced intercalation which is similar to (b), but the modified base and partner base are “expelled” from the interior of the DNA duolexes. 

Overall, some of these reactive PAH metabolites are mutagenic in human and bacterial cells, are carcinogenic in experimental animals, and are suspected to play a role in the etiology of many human cancers, especially lung-associated cancers.  Positive correlations between stable DNA adduct levels and susceptibility to cancer have been documented, and relevance of stable DNA adducts in human carcinogenesis has been widely recognized.

DNA repair and DNA Replication

The bay region diol epoxides of benzo[a]pyrene, e.g., the (+)- and   (-)-enantiomers of anti-B[a]PDE,  react with cellular DNA, predominantly with the exocyclic amino groups of guanine and adenine to form the stereoisomeric bulky B[a]P-dG N²-guanine adducts (Figure 2) and analogous B[a]P-dA N⁶-adenine adducts.  If these DNA adducts are not excised by normal cellular repair mechanisms, they can persist until DNA replication occurs and cause mutations if the replication is error-prone.  Multiple DNA mutations in critically important genes such as ras and p53 constitute genetic alterations that play key roles in the regulation of cell cycle control and cancer.  In B[a]PDE-treated HeLa and bronchial epithelial cells there is a remarkable preference for guanine adduct formation at CpG sites in codons 157, 248, and 273 of the p53 tumor suppressor gene, which are also the major mutational hot spots in human lung cancer associated with cigarette smoke. If a bulky lesion is successfully repaired by cellular nucleotide excision repair (NER) or other repair mechanisms, there will be no further consequences.  However, if the lesions escape repair and survive to the next round of DNA replication, faulty translesion bypass can occur giving rise to mutations and cancer.  Therefore, DNA repair and translesion synthesis catalyzed by polymerases are key factors that determine if a bulky lesion can give rise to mutations and ultimately to cancer. However, the molecular bases of these important mechanisms are still poorly understood.

Our Approach: From Basic Chemistry to Biology

Our research can be roughly divided into the following two inter-related and fully integrated experimental components:

(1)  Synthesis of completely defined DNA adducts derived from the reactions of PAH diol epoxide stereoisomers to form covalent adducts (Figure 2).  These days, however, we use automated DNA synthesis techniques based on phosphoramidite chemistry.  We generate modified oligonucleotides (short fragments of DNA with well defined nucleotide (base) composition and sequence, often resembling biologically important DNA sequences that occur in genomic DNA and that have been identified.  

(2) Chemical and Structural Origins of Biochemical and Biological impact.  Oligonucleotide duplexes bearing single adducts are then constructed for studies of DNA repair using human and bacterial repair enzymes, and DNA replication studies using the novel lesion (adduct) bypass polymerases in vitro. Site-directed mutagenesis experiments in cellular systems are carried out with collaborators (see list of publications).  The impact of such lesions on transcription are also being investigated in the laboratory of our collaborator Professor David Scicchitano in the NYU Biology Department.

Detailed Experimental Approaches and Objectives of our Research

We are interested in elucidating the detailed structural factors involved in the processing of bulky DNA adducts  derived from the binding of B[a]PDE and fjord PAH to DNA by nucleotide excision repair enzymes.  Our approach is unique in that the methods being used span a variety of experimental and computational chemistry approaches.   We can define the following flow chart that characterizes our approach:

(1) chemical synthesis of the site-specifically modified PAH lesions in oligonucleotides  (Figure 5) for detailed studies of DNA repair and translesion synthesis catalyzed by DNA polymerases in vitro and in vivo  

2) purification and structural analysis of the modified DNA that involves high performance liquid chromatography, mass spectrometry, gel electrophoresis, enzymatic processing of the DNA, circular dichroism and modern fluorescence methodologies (time-correlated single photon counting methods)

(3) Modern one-dimensional and multi-dimensional methods for determining the structural properties of the DNA adducts (PAH-oligonucleotide adducts).  In this area, we collaborate with Professor Dinshaw Patel of the Memorial Sloan-Kettering Cancer Center in New York, who is an expert in structural biology.

(4) Frontline computational chemistry techniques for analyzing the structural features and consequences of the PAH-DNA adducts.  In this area we collaborate with Professor Suse Broyde’s laboratory in the Biology Department. 

(6) Biochemical studies of enzyme activities in vitro employing gel electrophoresis coupled to modern imaging techniques for quantitative analysis.  Our present focus is on DNA repair (Figure 6) and DNA replication studies.

The main hypothesis in our projects is that alterations in the stereochemical properties of the DNA adducts and the DNA base sequence context in which the lesions are positioned can be utilized as tools to investigate the basic mechanisms and structure-function relationships in DNA repair and translesion DNA synthesis.  Our existing and substantial NMR structural data base, the continuously growing computer capabilities and greatly improved force fields and power, have made possible rapid advances in  the modeling of complex biomolecules.  These methods are now sufficiently robust to provide insights, on a molecular and structural level, into the mechanisms of translesion synthesis of bulky lesions, and their excision by DNA repair proteins.

Significance of Projects