POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.     

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.