Sequence specificity of single strand telomere DNA-binding proteins

Specificity in molecular recognition is the binding of the correct substrate and exclusion of incorrect though very similar looking substrates. What determines specificity? I have examined how Sterkiella nova telomere proteins distinguish cognate versus noncognate single stranded DNA sequences using approaches measuring both thermodynamic and kinetic performance. Grading subtle differences would be impractical with traditional methods, thus I developed of a new more sensitive method based on direct competition that reports nucleotide preference in the form of a high performance liquid chromatography chromatogram. The assay detected subtle degrees of specificity for S. nova telomere proteins, with nucleotide-binding sites ranging from neutral (binds all four bases with equal probability) to absolute (only accepts cognate base), and most sites intermediate (prefers cognate base but accepts alternatives), as indicated by character size: TTTTGGGGTTTTGGGG. Structures of the S. nova telomere end binding protein in complex with single strand DNA were re-examined in the light of recent measures of sequence specificity obtained with my new assay. Features from X-ray crystal structures that correlate strongly with high specificity include Watson-Crick like H-bonds between guanine bases and aspartate or glutamate amino acid residues. I further tested the role of these interactions by mutagenesis, measuring changes in specificity, binding affinities and kinetic performance for the resultant variant proteins. Mutational analysis reveals that bidentate H-bonds are important for sequence specific recognition. My study of association and dissociation rates leads to the conclusion that the rate with which protein releases substrate DNA is highly predictive of specificity performance. In the S. nova telomere system, specificity appears to have evolved by optimization/tuning of kinetic association and dissociation rates such that cognate DNA release from protein more slowly than do noncognate sequences. These findings likely reflect the reality that specificity operates in a dynamic and responsive (i.e., living) system and suggest important functional constraints that guide the course of telomere DNA and telomere protein co-evolution.