Abstract
Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5′-(TTAGGG)n-3′ double-stranded repeats, followed by up to several hundred bases of a 3′ single-stranded G-rich overhang. The G-rich overhang is bound by the shelterin component POT1 which interacts with TPP1, the component involved in telomerase recruitment. A previously published crystal structure of the POT1 N-terminal half bound to the high affinity telomeric ligand 5′-TTAGGGTTAG-3′ showed that the first six nucleotides, TTAGGG, are bound by the OB1 fold, while the adjacent OB2 binds the last four, TTAG. Here, we report two cryo-EM structures of full-length POT1 bound by the POT1-binding domain of TPP1. The structures differ in the relative orientation of the POT1 OB1 and OB2, suggesting that these two DNA-binding OB folds take up alternative conformations. Supporting DNA binding studies using telomeric ligands in which the OB1 and OB2 binding sites were spaced apart, show that POT1 binds with similar affinities to spaced or contiguous binding sites, suggesting plasticity in DNA binding and a role for the alternative conformations observed. A likely explanation is that the structural flexibility of POT1 enhances binding to the tandemly arranged telomeric repeats and hence increases telomere protection.
Highlights
Telomeres are protein-DNA complexes that protect chromosomes from degradation by preventing end-to-end fusion and countering the end replication problem [1–3]
Cryo-EM analyses of the purified and fully assembled shelterin complex (POT1, TPP1, TIN2, TRF2 and RAP1) were carried out on the unbound complex as well as the shelterin complex bound to a “model” telomere ligand (S1A Fig), containing both the appropriate double and single-stranded binding sites for the DNA-binding shelterin components protein 1 (POT1) and TRF2
Our hypothesis was supported during cryo-EM screening by the observation that very low concentrations (~0.05 mg/ml) of the POT1-TPP1-TIN2(1–354) sample used to prepare holey EM grids was consistently resulting in very high particle density
Summary
Telomeres are protein-DNA complexes that protect chromosomes from degradation by preventing end-to-end fusion and countering the end replication problem [1–3]. Mammalian telomeric DNA consists of 50-(TTAGGG)n-30 double-stranded repeats, followed by up to several hundred bases of single-stranded telomeric repeats forming a 30 G-rich overhang (Goverhang) [1–4]. Telomeres shorten with every cell division, and while this progressive shortening is generally considered a hallmark of aging, in stem and cancer cells it can be countered. Cryo-EM analysis of human POT1 and analysis, decision to publish, or preparation of the manuscript
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