Sequence-specific and 3'-end selective single-strand DNA binding by the Oxytricha nova telomere end binding protein alpha subunit.
Oxytricha nova telomere end binding protein (OnTEBP) specifically recognizes and caps single-strand (T(4)G(4))(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 alpha 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 alpha 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 alpha subunit. Addition of nucleotides to the 3'-end of the TTTTGGGG telomere repeat decreases the level of alpha 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 t(1)t(2)t(3)T(4)G(5)G(6)G(7)G(8) 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.
Classen
Scott
S
Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA. classen@uclink.berkely.edu
Lyons
Dan
D
Cech
Thomas R
TR
Schultz
Steve C
SC
D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D016054:DNA, Protozoan / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D016813:Oxytricha / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016615:Telomere / Q000235:genetics*; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D004277:DNA, Single-Stranded; D021122:Protein Subunits; D015800:Protozoan Proteins; D034501:Telomere-Binding Proteins