Human POT1 protects the telomeric ds-ss DNA junction by capping the 5′ end of the chromosome

Abstract

Protection of telomeres 1 (POT1) is the 3′ single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3–related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded–single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5′ end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a “POT-hole” surface, the mutation of which compromises junction protection in vitro and telomeric 5′-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a’s sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded–single-stranded junction.