Office of Biological and Environmental Research
DOE Low Dose Radiation Program Workshop V
Title: Molecular Energetics of Clustered Damage Sites
Authors and Institutions: Principal Investigator: Dr. Michel Dupuis (PNNL) Co-investigators: Professor John H. Miller (WSU Tri-Cities), Professor Robert D. Stewart (Purdue University), Dr. Maciej S. Gutowski (PNNL), Dr. Eric J. Ackerman (PNNL); Collaborators: Mr. Matt Hernst (WSU Tri-Cities), Dr. Vladimir A. Semenenko (Purdue University), Mr. Maciej Haranczyk (Gdansk University , Poland), Mr. Rafal A. Bachorz (Poznan University, Poland), and Ms. Iwona Dabkowska (Gdansk University, Poland).
The goal of this project is to provide critical information to help characterize clustered damage sites relative to singly damaged sites with respect to their susceptibility to DNA repair. The premise is that differences in base pairing rules and mutagenic properties of singly and multiply damaged DNA sites can be traced to local (<~10 base pairs) alterations in the energetic, structural, and dynamical properties of DNA. Aims are as follows: (1) to construct computational models of DNA lesions representative of those formed by endogenous processes and by low- and high linear energy transfer (LET) radiation; (2) to calculate the (free) energy of models of damaged and undamaged oligonucleotides using quantum chemical and molecular dynamics simulations; (3) to extract from the above computations sequence- and damage-specific (Boltzmann-based) probability distributions associated with key steps in the recognition and repair of singly and multiply damaged DNA sites as a link to higher-level Monte Carlo base and nucleotide excision repair models.
State-of-the-art computational chemistry models of quantum chemistry are used to characterize the structure, energetics, and spectroscopy of singly and multiply damaged (clustered) DNA sites. Abasic sites, strand breaks, and the oxidized purine 8-oxoG or 8-oxoA have been our focus to date. Using lesion-free trimers and pentamers as references, we predict the lesion “energies” (kcal/mol) to be: 0.2 for 8oA in AAC, -8.0 for apA in AAC, 25.3 for apT in AAC; 0.3 for 8oG in AGC, 15.6 for apG in AGC, 16.3 for apC in AGC; 2.3 for 8oG in AGT, 19.3 for apC in AGT; 1.9 for 8oG in ACA, 24.5 for apC in ACA. The results for 8-oxoG and 8-oxoA are consistent with Tmax measured by Plum et al., Biochemistry 34, 16148 (1995) and with other Nuclear Magnetic Resonance and x-ray data, that show very small structural reorganization induced by 8-oxoGC and 8-oxoAT. In contrast, Abasic sites cause substantial relaxation of the backbone, the details of which are dependent on sequence context. New inter-strand and intra-strand hydrogen bonds can be formed after removal of a nucleic acid base. Significant relocation of counterions may also be present. Current efforts using the same approach deal with multiply-damaged 8oG and apC in ACA, tymine glycol in CTA and ACTAG. The calculations are carried out on PNNL's Environmental Molecular Sciences Laboratory supercomputers.
The quantum calculations on small DNA fragments are also providing starting conformations and force-field parameters for classical molecular dynamics (MD) simulations of fully solvated and counterion balanced DNA oligonucleotides. Simulations are in progress for damaged bases, Abasic sites, and strand breaks, isolated and in combination. The goal of these calculations is to determine how close multiple lesions must be to have non-additive energetics and coupling of structural perturbations. These studies are based on the methodology of free energy perturbation calculations and theoretical mutations. Miller et al. (Journal of the American Chemical Society 2003) showed that perturbation of bending dynamics is a long-range effect of 8oG that may facilitate recognition. MD simulation is being used to determine if effects of this type lead to coupling between widely separated multiple lesions. Radiation-induced strand breaks are often accompanied by base loss. We have compared simple strand scission with strand breaks that include an Abasic site. When accompanied by a full nucleotide gap or a 3’ phosphoglycolate, backbone flexibility at the break is greatly increased relative to that induced by simple hydrolysis of a P-O bond. Since our MD simulations are carried out with fully solvated oligonucleotides, they provide insight into how lesions influence DNA hydration. Our results for 8oG show that the presence of water helps stabilize 8oG:C base pairs by decreasing the electrostatic repulsion between the O8 and backbone oxygen atoms in the same nucleotide.
Work is ongoing to relate calculated lesion energies to the probability of a polymerase inserting the correct nucleotide opposite a damaged nucleotide. These nucleotide insertion probabilities are one of the key inputs to a Monte Carlo excision repair model (Semenenko and Stewart 2004, supported by DOE grant DE-FG02-03ER63541, R.D. Stewart P.I., with partial support from this project).
Publications and Presentations
Haranczyk, M. Gutowski, M.S., Dupuis, M., Miller, J.H., Bachorz, R. and Dabkowska, I. (2005). Molecular energetics of clustered damage sites: DFT studies of structures and energetics of small oligonucleotides. Computational Biology and Chemistry (in preparation).
Miller, J.H., Aceves-Gaona, A., Ernst, M.B., Haranczyk, M., Gutowski, M.S., Vorpagel, E.R. and Dupuis, M. (2004). Molecular energetics of clustered damage sites. Radiation Research, special issue on the 3rd International Workshop on Space Radiation Research, 2004, Port Jefferson, New York.
Semenenko, V.A., Stewart, R.D., Ackerman, E.J. Monte Carlo simulation of the base and nucleotide excision repair of singly and multiply damaged DNA sites, Radiation Research.
Haranczyk, M., Bachorz, R., Dabkowska, I., Miller, J., Dupuis, M., Gutowski, M. Computational characterization of lesions in DNA In: ACS National Meeting, August, Philidelphia, Pennsylvania; AAAS Annual Meeting, February, Seattle, Washington; Nanoscale Science and Technology Workshop, September, The University of Washington, Seattle, Washington.
J.H. Miller , M. Dupuis, M. Gutowski, R.D. Stewart, V.A. Semenenko, E. Ackerman, M. Haranczyk, R.A. Bachorz, I. Dabkowska, (2004) Molecular Energetics of Clustered Damage Sites. In: 3d International Workshop on Space Radiation Research, May, Port Jefferson New York; DOE Low Dose Radiation Research Workshop IV, October 2003, Washington, D.C.
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