Binding of cations and proteins to DNA modulate both its structure and dynamics. In this grant application, we propose to investigate the role of cations and architectural proteins in binding to specific sites on DNA and their effects on the DNA structure. The research will focus particularly on cation binding to quadruplexes formed from telomere repeat sequences, the structure and sequence specific binding of cations to A-tract DNAs, and structure specific recognition of DNA by the architectural yeast HMG protein NHP6A. Multidimensional NMR spectroscopy will be used to investigate the structure of specific DNA sequences in the presence and absence of cations and proteins.
The specific aims are: 1. Investigate cation binding and hydrogen bonding to DNA quadruplexes. G-rich repeat sequences found in telomeres readily form quadruplex structures in the presence of appropriate size cations. We propose to investigate the effect of different cations on the hydrogen bond strengths and structures of DNA quadruplexes. 2. Investigate the structures of DNA duplexes containing A-tracts and how cation binding modulates the structures. DNA duplexes containing repeated runs of 4 or more AT base pairs without a TpA step in phase with the helical repeat are macroscopically bent. We propose to investigate the effect of monovalent and divalent cations on the structures of A-tract DNAs in solution. Accurate structures of the duplex DNAs will be determined by refining against residual dipolar couplings. 3. Investigate role of DNA structure in non-sequence specific HMG-box protein-DNA complexes. Architectural HMG box proteins like the yeast protein NHP6A modulate DNA structure and activity by inducing large bends in the DNA. These non-sequence specific proteins bind preferentially at distorted DNA structures such as 4-way junctions, intrastrand cisplatinum cross-links in duplex DNA, and in unmodified duplex DNA at sites which presumably have a pre- formed kink or bend. We propose to investigate the structure specific recognition of DNA by the yeast HMG box protein NHP6A to specific DNA duplexes and cis-platinated DNA. The effects of changes in the DNA sequences and mutations of specific residues in the protein will be investigated to reveal the structural determinants involved in DNA target selection. Conformational variability of DNA, and in particular DNA bending, play a significant role in basic cellular processes of transcriptional activation, DNA repair, replication, and recombination.
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