Occurrence, Dynamics, and Functions of Hoogsteen Base Pairs in DNA

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2021

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The structure and biological functions of the DNA double helix have been understood in the light of Watson-Crick (WC) base pairs (bps). However, bps can adopt an alternative base pairing conformation termed as the Hoogsteen (HG) bps in double-stranded DNA. The WC bps have been shown to exist in dynamic equilibrium with short-lived low-populated HG bps in vitro. This thesis studies the modulation of the WC-HG equilibrium on DNA recognition, the potential role of HG bps in DNA damage, and also develops new sequencing approaches to map the HG bps in vivo.To study how the WC / HG dynamics was modulated upon recognition of duplex DNA by small molecule drugs, we applied NMR relaxation dispersion experiments and molecular dynamics simulations to a bisintercalator echinomycin and a monointercalator actinomycin D. In both cases, DNA recognition resulted in the quenching of HG dynamics at bps involved in intermolecular base-specific hydrogen bonds. In the case of echinomycin, the HG population increased 10-fold for bps flanking the chromophore most likely due to intermolecular stacking interactions, whereas actinomycin D minimally affected HG dynamics at other sites. The results revealed that modulation of HG dynamics at binding interfaces could be a general phenomenon with important implications for DNA-ligand and DNA-protein recognition. To explore the thermodynamic propensities of DNA to adopt minor conformations such as HG bps, a simple, fast, and cost-effective approach called “delta-melt” was developed that combining optical melting experiments with chemical modifications and mutations. With this approach, we obtained unique insights into the thermodynamic cooperativity in HG formation at adjacent bps. This finding was verified by NMR experiments and provides an explanation for the frequent observation of tandem HG bps in crystal structures of DNA duplexes containing HG bps. Because HG bps expose the WC faces of the purine nucleobases to the solvent, we reasoned that they could potentially lead to damage to the WC faces of purines. To test this hypothesis, we developed a biochemical assay that combines chemical probing, dot-blot, and primer extension to assess HG-mediated damage to the WC faces of nucleobases. The results indicated that the reactivity to dimethyl sulfate (DMS) of adenine-N1 in HG A-T bps is ~2-4 fold higher than that in WC A-T bps, suggesting potential roles of HG bps in cytotoxic damage induction in double-stranded DNA. The unique DMS reactivity signature of HG bps provided a means to discriminate HG from WC bps. By extending this DMS chemical probing method and combining it with the next-generation sequencing (NGS) technology, we developed a new DNA sequencing method, “2dDMS-seq” to comprehensively map the non-canonical structures of genomic DNA in living cells strand-specifically and at single nucleotide resolution. The method utilizes both N1-methylated adenine (m1A) and N3-methylated adenine (m3A) produced by DMS treatment as probes for non-canonical DNA structures. Applied to the yeast Saccharomyces cerevisiae, this approach probed single-stranded DNA (ssDNA) regions, nucleosome (NCP) positioning, transcription factors (TF)-binding sites, autonomously replicating sequences (ARS), and potential HG A-T bps, and thus provided in-depth insights into the structure, accessibility, damage, repair, and protein-binding landscapes of genomic DNA in vivo.

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XU, YU (2021). Occurrence, Dynamics, and Functions of Hoogsteen Base Pairs in DNA. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/23010.

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