Mutations drive evolution, account for genetic variants in the population, and are the primary cause of cancer and other genetic disorders. Yet our molecular understanding of the biochemical processes that cause mutations remains rudimentary. For most mutational processes, we do not understand why the mutational probabilities vary by many orders of magnitude depending on the type of base substitution and sequence context. Most mutational patterns cannot be explained by the 1D sequence or 3D structural characteristics of the DNA motif in which they are found. While mutational processes due to exogenous sources (e.g. UV, smoking) have been described extensively, studies increasingly point to DNA replicative errors as an important and potentially dominant source of disease-causing mutations. However, the molecular mechanisms that underlie DNA replicative errors and their contributions to oncogenesis are not fully understood. In addition, over half of the mutational processes identified in human cancers have unknown biochemical origins. The main hypothesis in this proposal is that DNA dynamics that alter the mode of base pairing is a major driver of mutational processes. The project will experimentally characterize sequence and mismatch-dependent DNA base pair dynamics with unprecedented breadth and depth, and generate conformational propensities describing the sequence-specific probabilities of forming alternative mutagenic conformations. This knowledge will be used to develop a predictive understanding of replication errors generated by human polymerase ?, one of two polymerases tasked with eukaryotic nuclear DNA replication. The critical and necessary technological innovation is the development of breakthrough techniques for measuring DNA structural dynamics in high throughput, enabling studies of over hundreds and in some cases thousands of sequence variants.
Aim 1 will determine the propensities for various mismatches to form Watson-Crick like conformations, measure the signatures of replicative error for proofreading deficient human polymerase ?, and advance a predictive model for sequence- and mismatch- dependent nucleotide misincorporation.
Aim 2 will determine the propensities to sample unpaired conformations, measure the signatures of replicative error for proofreading proficient human polymerase ?, and advance a predictive model for sequence- and mismatch-dependent replicative errors.
Aim 3 will determine propensities to form Hoogsteen base pairs, and uncover mutational processes driven by Hoogsteen-mediated damage. By developing a deep and predictive understanding of DNA replication infidelity and damage, this work will help illuminate fundamental processes that drive evolution and oncogenesis while also providing a conceptual framework and experimental tools that can help catalyze the discovery and characterization of other mutagenic and biochemical processes driven by DNA dynamics.

Public Health Relevance

The proposed research is relevant to public health because understanding the basis for mutational processes is expected to deepen our understanding of cancer and other genetic disorders and to give rise to improvements in our ability to predict, diagnose, and ultimately treat these genetic diseases. Upon conclusion of the proposed studies, we will have a better understanding regarding the mechanisms that underlie mutations due to DNA replicative errors as well as the role of DNA structural dynamics in mutagenic damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM089846-11
Application #
10124402
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sakalian, Michael
Project Start
2010-09-15
Project End
2023-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
11
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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Shi, Honglue; Clay, Mary C; Rangadurai, Atul et al. (2018) Atomic structures of excited state A-T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations. J Biomol NMR 70:229-244
Kimsey, Isaac J; Szymanski, Eric S; Zahurancik, Walter J et al. (2018) Dynamic basis for dG•dT misincorporation via tautomerization and ionization. Nature 554:195-201
Stelling, Allison L; Xu, Yu; Zhou, Huiqing et al. (2017) Robust IR-based detection of stable and fractionally populated G-C+ and A-T Hoogsteen base pairs in duplex DNA. FEBS Lett 591:1770-1784
Szymanski, Eric S; Kimsey, Isaac J; Al-Hashimi, Hashim M (2017) Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson-Crick-like Base Pairs. J Am Chem Soc 139:4326-4329
Zhou, Huiqing; Kimsey, Isaac J; Nikolova, Evgenia N et al. (2016) m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs. Nat Struct Mol Biol 23:803-10
Kimsey, Isaac J; Petzold, Katja; Sathyamoorthy, Bharathwaj et al. (2015) Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes. Nature 519:315-20
Salmon, Loïc; Giamba?u, George M; Nikolova, Evgenia N et al. (2015) Modulating RNA Alignment Using Directional Dynamic Kinks: Application in Determining an Atomic-Resolution Ensemble for a Hairpin using NMR Residual Dipolar Couplings. J Am Chem Soc 137:12954-65
Ficici, Emel; Andricioaei, Ioan (2015) On the Possibility of Facilitated Diffusion of Dendrimers Along DNA. J Phys Chem B 119:6894-904
Mentes, Ahmet; Florescu, Ana Maria; Brunk, Elizabeth et al. (2015) Free-energy landscape and characteristic forces for the initiation of DNA unzipping. Biophys J 108:1727-1738

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