Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 [A] resolution. The structure reveals a CST and a pol-/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.