The genome is encoded within double-stranded DNA. The properties and interactions of this important macromolecule are dominated by its polyelectrolyte character. For example, the high negative charge density of DNA causes counterion condensation, a phenomenon that has implications for all protein/DNA interactions. Its high negative charge density of DNA also causes DNA to behave as a relatively stiff, worm-like polymer in dilute solution. In contrast to its behavior as a naked polymer, DNA can be highly bent in complexes with certain proteins. Such DNA bending is believed to be critical for DNA packaging and for biological functions such as transcriptional regulation. We are interested in the role of electrostatic (charge-charge) interactions in DNA bending by proteins and ions. In particular, we have been testing the hypothesis that laterally-asymmetric phosphate neutralization by cationic proteins can induce DNA to collapse toward its neutralized surface because of unbalanced interphosphate repulsions within the double helix. Results of our previous studies of chemically-modified DNA strands and charge variants of DNA binding proteins tend to support this simple hypothesis. The present proposal seeks to refine and extend this model, ultimately placing our studies of DNA bending in a more biological context. Four specific aims will be undertaken: First, we will attempt to corroborate our prior observation that DNA bending results when derivatives of the yeast bZIP DNA binding protein GCN4 contain cationic or anionic amino acid substitutions near their basic regions. Second, we will determine the role of asymmetric phosphate charge neutralization in DNA bending by the E. coli CAP protein and the mammalian histone octamer. Third, we will tether multivalent cations or anions at a single DNA site and observe effects on DNA bending. Fourth, we will study the importance of DNA bending in a biological context by exploring how the flexibility of DNA affects transcription activation using a eukaryotic transcription system.
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