Single- and double-stranded DNA binding proteins act in concert to conserve a telomeric DNA core sequence

We found that the Saccharomyces castellii protein Rap1, a negative regulator of telomere length, binds a 12-mer minimal binding site (MBS) within the double-stranded telomeric DNA. The sequence specificity is dependent on the interaction with two 5 nucleotide motifs, having a 6 nucleotide centre-to-centre spacing. The isolated DNA-binding domain binds the same MBS and retains the same motif binding characteristics as the full-length Rap1 protein. However, it shows some deviations in the degree of sequence-specific dependence in some nucleotide positions. Intriguingly, the positions of most importance for the sequence-specific binding of the full-length Rap1 protein coincide with 3 of the 4 nucleotides utilized by the 3' overhang binding protein Cdc13. These nucleotides are very well conserved within the otherwise highly divergent telomeric sequences of yeasts.Rap1 and Cdc13 are two very distinct types of DNA-binding proteins with highly separate functions. They interact with the double-stranded vs. the single-stranded telomeric DNA via significantly different types of DNA-binding domain structures. However, we show that they are dependent on coinciding nucleotide positions for their sequence-specific binding to telomeric sequences. Thus, we conclude that during the molecular evolution they act together to preserve a core sequence of the telomeric DNA.The ends of eukaryotic chromosomes form specialized chromatin structures called telomeres, which protect the chromosome ends from being degraded or recognized as double-strand breaks by the DNA damage response pathway. The assembly of the protective telomere cap structure is nucleated by the sequence-specific proteins binding to the double-stranded telomeric DNA and the single-stranded 3'-overhang, respectively. In addition to the shielding and protective role, the telomere binding proteins take part in a molecular mechanism which regulates the telomere to a species-specific length. In budding yeast, telomere length hom