Sequence-Specific and 3‘-End Selective Single-Strand DNA Binding by the Oxytricha nova Telomere End Binding Protein α Subunit

Oxytricha nova telomere end binding protein (OnTEBP) specifically recognizes and caps single-strand (T4G4)2 telomeric DNA at the very 3‘-ends of O. nova macronuclear chromosomes. The discovery of proteins homologous to the N-terminal domain of the OnTEBP α subunit in Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens suggests that related proteins are widely distributed in eukaryotes. Previously reported crystal structures of the ssDNA binding domain of the OnTEBP α subunit both uncomplexed and complexed with telomeric ssDNA have suggested specific mechanisms for sequence-specific and 3‘-end selective recognition of the single-strand telomeric DNA. We now describe comparative binding studies of ssDNA recognition by the N-terminal domain of the OnTEBP α subunit. Addition of nucleotides to the 3‘-end of the TTTTGGGG telomere repeat decreases the level of α binding by up to 7-fold, revealing a modest specificity for a 3‘-terminus relative to an internal DNA binding site. Nucleotide substitutions at specific positions within the t1t2t3T4G5G6 G 7G8 repeat show that base substitutions at some sites do not substantially decrease the binding affinity (20-fold decrease for the uppercase bold letter). Comparison of the structural and binding data provides unique insights into the ways in which proteins recognize and bind single-stranded DNA.