Our basic understanding of how DNA carries out its biological function has been based on the Watson-Crick double helix as the dominant functional form of duplex DNA. However, Watson-Crick base-pairs cannot explain many fundamental biochemical aspects of duplex DNA, including how proteins recognize DNA with high sequence-specificity; how Watson-Crick faces of nucleotide base-pairs can become appreciably solvent exposed and prone to chemical damage; how damaged base-pairs can be stably accommodated in DNA and recognized by repair enzymes; and how errors arise during replication, transcription, and translation. The main hypothesis in this proposal is that canonical Watson-Crick base-pairs and non-canonical mispairs can transiently adopt alternative, higher energy, and sparsely populated conformations that are difficult to detect and characterize by biophysical methods, and that these transient alternative base-pairs provide a new layer of structural and dynamic complexity that is employed to drive many important DNA functions.
Aim 1 will develop methods for identifying and experimentally characterizing Hoogsteen base-pairs in X-ray structures of DNA that may have been improperly modeled as Watson-Crick base-pairs.
This Aim will also test the hypothesis that Hoogsteen base-pairs play important roles in maintaining genome stability in structurally stressed environments and in sequence-specific DNA recognition by proteins.
Aim 2 will test the hypothesis that Hoogsteen base-pairs provide a basis for exposing Watson-Crick faces of nucleotide bases for sequence-specific alkylation damage. It will also test the hypothesis that damaged bases can be stably accommodated in DNA as Hoogsteen base-pairs that induce DNA bending and play functional roles in recognition by repair enzymes.
Aim 3 will develop methods to characterize transient Watson-Crick-like mispairs that are stabilized by rare tautomeric and anionic bases. We will test the hypothesis that anionic Watson-Crick-like G-T mispairs provide the basic mechanisms for spontaneous and damaged-induced G-T misincorporation during DNA replication. The proposed fundamental studies of DNA structure and dynamics will redefine our view of the iconic DNA double helix, and uncover a rich layer of mechanistic complexity hidden in unconventional base-pairs that have so far proven difficult to capture and characterize at atomic resolution.
This project will characterize dynamic DNA base-pairs that play essential roles in sequence-specific DNA-protein recognition, genome stability, damage induction, accommodation and repair, and spontaneous and damaged-induced mutations. The research is expected to significantly improve our understanding of the mechanisms that lead to diseases such as cancer as well as expose new structural forms of DNA for targeting with the use of small molecule therapeutics.
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