Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state

Abstract

The CST–Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST–Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST–Polα/primase complex map to protein–protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.

PDBe and PDBe‐KB: Providing high‐quality, up‐to‐date and integrated resources of macromolecular structures to support basic and applied research and education

“…The advent of these advanced computationally predicted models disrupted several fields in the life sciences, from structure‐based drug discovery and bioinformatics analysis to software development and structure determination efforts. 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 Researchers regularly use AlphaFold models to solve protein structures using their (sometimes old) experimental data.…”

Reconstitution of a telomeric replicon organized by CST

“…In support of this idea, the three genes for the CST components rose to the very top in CRISPR-Cas9 screens for genes important for growth of cancer cell lines with short telomeres 37 . On the basis of the work presented here, the ssDNA-binding site in the CTC1 subunit 19 or Polα-primase-binding sites in all three CST subunits 32,38 seem particularly attractive targets for interfering with telomere end replication.…”

“…c, Reactions under the conditions of ref. 38 , with 50-fold higher DNA template concentrations (5 μM). For the 15xTEL template, the difference in activity was greater than 10-fold; compare the two 10 nM reactions, lanes 7 and 9;…”

CST/Polα/primase-mediated fill-in synthesis at DSBs

“…Composite models were generated from cryo-EM structures (PDB-6W6W [ 25 ], maps (EMD -21,563 46 ), and AlphaFold models of individual subunits (AF- Q54WQ3/Q9H668/Q86WV5 [ 53 , 54 ]. e , Cryo-EM structure of CST/Polα/primase in the recruitment complex conformation [ 48 ]. The model is scaled and rotated about CST relative to (d) as indicated.…”

“…The telomere-specific functions of CST rely on its recruitment by the TPP1 and POT1 subunits of shelterin ( Figure 2c ), which, like CST, are needed for reconstituting the 5’-ended C-strand after DNA replication [ 32 , 34 , 38 , 41–47 ]. Cryo-EM studies showed that CST/Polα/primase is recruited to telomeres in a compact, auto-inhibited state (recruitment complex (RC); Figure 2(c-e) ), where the POLA1 active site is occluded [ 48 ]. In ways that are not understood, the complex then transitions into an extended, active state, where the POLA1 catalytic core is accessible for catalysis [ 49 ] (pre-initiation complex (PIC); Figure 2f ).…”