Mammalian shelterin proteins POT1 and TPP1 form a stable heterodimer that protects chromosome ends and regulates telomerase-mediated telomere extension.However,how POT1 interacts with TPP1 to form a heterodimer remains unknown.Here we present the crystal structure of the C-terminal portion of human POT1(POT1C)complexed with the POT1-bindingmotif(PBM)of TPP1 at a resolution of 2.1 (A).The structure shows that POT1C contains two domains,an OB-fold and a Holliday Junction Resolvase-like domain.Both domains are essential for the binding with TPP1.We identified several missense mutations in human cancers that disrupt the POT1C-TPP1 interaction,resulting in POT1 instability.POT1C mutants that bind TPP1 localize to telomeres but fail to repress a DNA damage response and inappropriate repair by ANHEJ.Our results reveal that POT1 C-terminus is essential to prevent initiation of genome instability permissive for tumorigenesis.