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

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, 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 humansMight be related to malignancies of the colon and breast in humans

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.

H2O X X

N-CH3

G6

PhIP-DNA adduct

DNA Control

(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.

Che

mic

al S

hift

Diff

eren

ceC

ontro

l-PhI

P-D

NA

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.

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.

C5

G6*

C7

G18

C17

G16

C5

G6*

C7

G18

G16

C17

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*§.

• 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)