| The recognition of single-stranded nucleic acids plays an important role in many cellular processes, including DNA replication, transcription, DNA repair and recombination, translation, RNA processing, and telomere regulation. We have used the essential S. cerevisiae protein Cdc13 as a model for gaining a detailed structural and biochemical understanding of how single-stranded DNA can be recognized with both high affinity and specificity.; Cdc13 performs at least two essential functions. It caps telomeres by protecting the C-rich strand from degradation and facilitates telomeric replication by regulating the specialized reverse transcriptase telomerase. In vitro it has been shown to bind single-stranded yeast telomeric DNA of the consensus sequence G2–3(TG)1–6 with high affinity and specificity (0.3 nM). An independent, modular DNA-binding domain (DBD) had been previously identified in the laboratory of Vicki Lundblad. We have used trypsin proteolysis and MALDI mass spectrometry to further refine this domain to its minimal necessary and sufficient construct. This domain (aa 497–694) binds to the minimal DNA oligomer (dGTGTGGGTGTG) with much higher affinity than the full-length protein, and it exhibits photoaffinity-crosslinked species along the entire length of the DNA.; The high-resolution solution structure of this protem/DNA complex was solved by heteronuclear NMR in collaboration with Rachel Mitton-Fry. Although not previously detected due to low sequence similarity, this domain was found to be a member of the oligosaccharide-oligonucleotide (OB) fold superfamily. It is thus a structural and functional homolog of the other known telomere-binding proteins O. nova TEBP, S. pombe Pot1, and human Pot1. A combination of chemical and isotopic substitutions in the DNA allowed for full resonance assignment. Select/filter NMR experiments were used to identify NOE contacts between the protein and the DNA. The DNA in the complex is in a fully extended form with numerous contacts to hydrophobic, aromatic, and basic residues in the protein. A complete alanine scan of the protein sidechains at the interface was performed to determine their thermodynamic contributions to binding. Overall, these hydrophobic and aromatic residues contributed significantly to binding, with a region corresponding to the canonical OB-fold binding surface having the greatest effect in recognizing the 5 ′ GTGT of the DNA. This binding interface is expanded in the Cdc13 DBD by addition of a large 30-amino acid loop containing several tyrosine residues that are also important for recognition. |
