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

• Published in: Biochemistry (Easton) Vol. 42; no. 31; pp. 9269 - 9277
• Main Authors: Classen, Scott, Lyons, Dan, Cech, Thomas R, Schultz, Steve C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.08.2003
• Subjects: Animals; Binding Sites; Crystallography, X-Ray; Dimerization; DNA, Protozoan - genetics; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Electrophoretic Mobility Shift Assay; Oxytricha - chemistry; Oxytricha - genetics; Protein Conformation; Protein Subunits; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Substrate Specificity; Telomere - genetics; Telomere-Binding Proteins - chemistry; Telomere-Binding Proteins - genetics; Telomere-Binding Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0273718

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 (<2-fold for lowercase letters), while substitutions at other sites dramatically reduce 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.