1. The NMR Structure of the PhIP-DNA Adduct Karen Brown, Elizabeth A. Gunther, V. V. Krishnan, Kenneth W. Turteltaub and Monique Cosman, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore CA 9455, USA Brian E. Hingerty, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA Suse Broyde, Biology Dept., New York University, New York, NY 10003, USA

2. Felton, Knize, Shen, Lewis, Andressen, Happe, Hatch (1986) The isolation and identification of a new mutagen from fried ground beef: 2-amino-1-methyl-6 phenylimidazo[4,5-b)pyridine (PhIP) Carcinogenesis, 7, 1081-1086. The most abundant of the mutagenic heterocyclic amines found in cooked meats and fish, Might be related to malignancies of the colon and breast in humans

3. Possible mechanism of formation of C8-guanyl DNA adducts from activated heterocyclic amines. From Guengerich et al. (1995) in Heterocyclic Amines in Cooked Foods: Possible Human Carcinogens (Ed. Adamson, et al) Princeton Scientic Publishing Co., Inc. Princeton, NJ What are the structural alterations induced in the DNA and how do these alterations affect interactions with cellular proteins that may lead to disease? DNA damage can lead to mutations - a heritable change in the DNA sequence that can have devastating consequences, such as genetic disease and cancer.

4. H2OH2O XX N-CH 3 G6 PhIP-DNA adduct DNA Control

5. (1) Two conformations are present for the central three DNA base pairs and PhIP ligand (2) In the major conformation, the modified G6 exhibits loss of stacking interactions within the helix. Its partner C17 is also displaced outside the helix. The flanking G16 and G18 residues and PhIP ligand are stacking with one another. The PhIP ligand exhibits NOEs with DNA protons in both the major and minor groove -> intercalating. (3) The DNA in the minor conformer is relatively unperturbed and the PhIP ligand is most likely located external to the helix in the major groove. Chemical Shift Difference Control-PhIP-DNA

6. Computational Methods Restrained conformational searches were performed using the program DUPLEX, a molecular mechanics program for nucleic acids that performs energy minimization in the reduced variable domain of torsion angle space. This method allows solution of the structure starting far from the minimum.

7. Combined NMR Energy Minimization Olson and Srinivasan Energy Evaluation Van der Waals terms Coulombic terms Distance dependent dielectric function Hydrogen bond parameters and restraints Anomeric electron repulsion terms Quadratic restraints which penalize the total function if the calculated NMR NOE distance is out of the experimental range Joint minimization and search using the combined theoretical and experimental function.

8. C5 G6* C7 G18 C17 G16 C5 G6* C7 G18 G16 C17

9. Legend: Heterocyclic amines, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (in magenta), are mutagens and carcinogens that are formed during the cooking of meats. They can covalently bind to DNA (left) and dramatically distort its structure (right). If the damaged DNA is not repaired, tumorgenesis may result. (Protein Data Bank access code 1HZ0) Solution structure of the 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA Karen Brown*, Brian E. Hingerty †, Elizabeth A. Guenther*, V. V. Krishnan* Suse Broyde ‡, Kenneth W. Turteltaub* and Monique Cosman* §.

11. Lihua Wang (NYU) for helical parameters US DOE contract W-7405-ENG-48 and NIH CA55861 and RR13461 for LLNL US DOE contract DE-AC0500-OR22725 for ORNL US DOE DE-FG0290ER60931 and NIH CA75449 to New York University US DOE National Energy Research Scientific Computing Center at Lawrence Berkeley Nat. Lab. NSF Partnership for Advanced Computational Infrastructure (UC San Diego)