Arginine-rich motif peptides as tools for understanding single-stranded DNA recognition

Although DNA is generally found in the double-stranded form, many of the important events that happen to it occur while it is single-stranded. In many of these cases, it is expected that the recognition of specific single-stranded DNA (ssDNA) sequences by proteins will be essential for the fulfillment of ssDNA function. Very little is known about the mechanisms of ssDNA recognition by proteins, but much of what is known has come from structural studies on the specific binding of linear, unstructured DNA sequences by what are conventionally thought of as "RNA-binding domains". Given the requirement that any ssDNA sequence in the cell must compete with the duplex form for its existence, it is likely that stable secondary and tertiary structures will also be important for ssDNA recognition. By analogy to linear ssDNA recognition, the study of protein domains specific for structured RNA should prove valuable for understanding how structured ssDNA is recognized.; This thesis describes the use of the arginine-rich motif (ARM) family of RNA binding proteins to understand ssDNA recognition. Initially, we show that, while the affinity of at least one ARM:RNA interaction depends strongly on features of the A-form RNA helix, this dependence is localized and that multiple energetically critical protein:RNA contacts appear to be viable in the B-form helix geometry that is characteristic of DNA. We expand on this by using in vitro selection to identify high affinity ssDNA ligands to the ARM peptide from the HIV Rev protein. We show that Rev can bind ssDNA with affinities and specificities that compare favorably to its ability to recognize RNA. We also characterize a mechanism for the recognition of branched nucleic acids in which an aromatic amino acid sidechain appears to stack on the end of one DNA helix in the context of a 3-helix junction structure that emerged from our selection. Finally, we identify a simple interaction motif, a 5' G•T/CG basestep that is recognized by two arginine separated by one turn of an alpha-helix, which should facilitate specific ssDNA binding by a variety of nucleic acid binding proteins to ssDNA in a range of structural contexts.