Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

CST–Polα/Primase maintains telomeres through fill-in synthesis of the C-rich telomeric DNA. We report cryo-EM structures that reveal how human CST is recruited to telomeres by the shelterin subunits POT1 and TPP1. CST–POT1/TPP1 is formed through interactions between POT1 and the Ctc1 subunit of CST. Coats plus syndrome mutations map to the POT1–Ctc1 interface, providing mechanistic insights into this disease. CST–POT1/TPP1 is compatible with the previously reported inactive recruitment complex of CST–Polα/Primase but not with the distinct conformation of active CST–Polα/Primase. We propose that shelterin both recruits and regulates CST–Polα/Primase. Structural and biochemical data indicate that this regulation involves phosphorylation of POT1, which promotes CST–POT1/TPP1 interaction and recruitment, whereas POT1 dephosphorylation releases CST–Polα/Primase for fill-in synthesis.One-Sentence SummaryCryo-EM structures reveal how telomere maintenance factors are recruited and regulated by the shelterin complex.