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Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA.
M K Raghuraman1, C J Dunn, B J Hicke
1Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, Boulder, CO 80309-0215.
Nucleic Acids Research
|June 12, 1989
Summary
The Oxytricha nova telomere protein specifically binds to (T4G4)n DNA sequences, protecting the 3' end. This binding is crucial for telomere maintenance and suggests a revised model for telomere synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Telomeres protect chromosome ends from degradation.
- The telomere binding protein in Oxytricha nova is essential for telomere maintenance.
- Previous studies suggested the protein binds to the 3'-terminal single-stranded (T4G4)2 tail of macronuclear DNA.
Purpose of the Study:
- To investigate the binding properties of the Oxytricha nova telomere binding protein using synthetic oligodeoxynucleotides.
- To determine the specific DNA sequences recognized by the telomere protein.
- To elucidate the molecular basis of telomere-protein interactions.
Main Methods:
- In vitro binding assays using synthetic oligodeoxynucleotides with varying (T4G4)n and C4A4 sequences.
- Nondenaturing gel electrophoresis to resolve protein-DNA complexes.
- Methylation protection assays to assess DNA binding specificity.
- Site-directed mutagenesis to probe the role of specific DNA bases and modifications.
Main Results:
- The telomere protein specifically binds to single-stranded oligonucleotides with the sequence (T4G4)n (n≥2) at the 3' end, protecting it from probes.
- Binding specificity was confirmed by the formation of three distinct protein-DNA complexes in gel electrophoresis.
- Oligonucleotides with C4A4 repeats were also recognized, forming a single complex.
- The dissociation constant for (T4G4)4 was approximately 19 nM, with significantly lower affinity for macronuclear DNA.
- Transversions in the 3' tail (T to A or G to C) prevented binding, while deoxyuridine substitution did not affect binding.
Conclusions:
- The Oxytricha nova telomere binding protein exhibits high specificity for (T4G4)n sequences, crucial for telomere protection.
- The protein's binding is not solely dependent on the hydrophobic 5-methyl group of thymine.
- These findings support a revised model for telomere synthesis in Oxytricha, highlighting the protein's role in recognizing and binding specific telomeric DNA structures.