Abstract
BackgroundTelomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. However, the telomeric sequences are highly diverged among yeast species. During the evolution this high rate of divergency presents a challenge for the sequence recognition of the telomere-binding proteins.ResultsWe 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.ConclusionsRap1 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.
Highlights
Telomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions
To be able to analyze the inherent DNA binding properties of the DNA-binding domain (DBD), the DBD coding region of the scasRAP1 gene was isolated by PCR amplification of the DNA region corresponding to amino acids 336-582 [2]
It was cloned, expressed in E. coli BL21 cells, and the purified scasRap1-DBD protein was run on SDS-PAGE to confirm the expected size (Additional file 1, Figure S1A)
Summary
Telomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. 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. When the telomeric DNA is extended and more Rap proteins can bind, the telomere is suggested to fold into a higher-order complex which is non-accessible for telomerase [10]. The molecular details for this regulatory mechanism are not fully unravelled
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