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

Abstract

DNA double-strand breaks (DSBs) pose a major threat to the genome, so the efficient repair of such breaks is essential. DSB processing and repair is affected by 53BP1, which has been proposed to determine repair pathway choice and/or promote repair fidelity. 53BP1 and its downstream effectors, RIF1 and shieldin, control 3' overhang length, and the mechanism has been a topic of intensive research. Here, we highlight recent evidence that 3' overhang control by 53BP1 occurs through fill-in synthesis of resected DSBs by CST/Polα/primase. We focus on the crucial role of fill-in synthesis in BRCA1-deficient cells treated with PARPi and discuss the notion of fill-in synthesis in other specialized settings and in the repair of random DSBs. We argue that - in addition to other determinants - repair pathway choice may be influenced by the DNA sequence at the break which can impact CST binding and therefore the deployment of Polα/primase fill-in.

Keywords: 53BP1; CST; DSB repair; Polα/primase.

Figure 1.

The 53BP1 pathway controls DNA end structure at DSBs. a, Schematic of the DNA end structures acted upon by the primary DSB repair pathways. DSB: double-strand break; cNHEJ: classical non-homologous end-joining; HDR: homology-directed repair; SSA: single-strand annealing. b, Top, overview of 53BP1, RIF1, shieldin, and CST/Polα/primase interactions. Bottom, Two models for how the 53BP1 pathway limits 3’ overhang length. c, Three context-dependent repair reactions where 53BP1 activity has been studied. Check marks indicate a requirement for a 53BP1 downstream effector or function. Question marks and reduced opacity cartoons denote uncertainties based on the current data.

Figure 2.

CST and CST/Polα/primase structure and function. a, Domain schematics for CST and Polα/primase subunits. OB: oligosaccharide/oligonucleotide-binding fold; wH: winged helix-turn-helix; 3 H: 3-helix bundle; N: POLA1 flexible N-terminal region; NTD: N-terminal domain; EXO: exonuclease domain, inactive; CTD: C-terminal domain; CRL: CTC1-Recognition Loop; PDE: phosphodiesterase domain. Colors from domain schematics are consistent in all figures. CP mutations in fill-in components are indicated with markers. Colors indicate mutants reported to disrupt Polα/primase association (red), ssDNA-binding (cyan), or CST complex formation (orange). Black markers represent mutants with other or uncharacterized defects. b, Summary of CST substrates and reported affinities (order of magnitude). c, Cartoon summary of CST and CST/Polα/primase cellular functions. RC: recruitment complex; PIC: pre-initiation complex. d, Structural models of CST in ssDNA-bound (top) and apo (bottom) conformations. Composite models were generated from cryo-EM structures (PDB-6W6W [25], maps (EMD -21,563⁴⁶), 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. f, Cryo-EM structure of CST/Polα/primase in the pre-initiation complex conformation [49]. The model is rotated about CST relative to (e) as indicated.

Figure 3.

CST-shieldin interactions. a, Domain schematics for CST and shieldin subunits, with interacting regions mapped in gray. Abbreviations as in Figure 2A. EIF4E-l: eukaryotic translation initiation factor 4E-like. HORMA: HOP1, REV7, MAD2-like. SHLD3 interacts with two copies of REV7 in a 1:2 stoichiometry [67]. b, Composite structural model of CST-shieldin complex generated from the cryo-EM structure of ssDNA-bound CST (PDB-6W6W [46]) superposed with AlphaFold-multimer models [61,62] (default settings, max_template_date=2022-07-01, AMBER relaxed) of CTC1-SHLD1, SHLD1-SHLD2-SHLD3, and the crystal structure of SHLD2-C-REV7-O-REV7-SHLD3 (O-REV7: open conformation; C-REV7: closed conformation; PDB-6KTO [67]). Flexible, unstructured regions predicted with low confidence (pLDDT < 30) are shown with dashed lines. Models were aligned in PyMOL (Schrödinger) and visualized in ChimeraX [68]. c, Top, zoom-in of the CTC1-SHLD1 interface with interacting residues labeled. Residues highlighted in bold face (L18, D19, L20, and P21) are deleted in the SHLD1D mutant. Bottom, SHLD1 N-terminus sequence, adapted from [7]. Asterisks indicate residues which, upon mutation, weaken the interaction between CTC1 and SHLD1. Red asterisks indicate loss of interaction due to a single amino acid substitution or deletion. Residues deleted in SHLD1D and the alpha helix (residues 23-32) are indicated. d, Predicted aligned error (PAE) plots of the top six ranked CTC1-SHLD1 Alphafold-multimer models. Domain schematics the same as in a. Green arrows indicate high-confidence in the position prediction of SHLD1 N-terminus relative to CTC1.

Figure 4.

CST binding to telomeric and CSR sequences. Representative gels from EMSAs measuring CST binding to 0.1 nM radioactive ³²P 5’ end-labeled substrates: AT rich (TATATA)₃, Telomeric (GGTTAG)₃, mouse IgA locus G-rich sequence (AGAGGAGGAGAGGAGAGG), and mouse IgA locus C-rich sequence (CCTCTCCTCTCCTCCTCT). A serial dilution of purified CST [48] (shown left) was incubated at room temperature for 30 min with labeled substrate in binding buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM MgCl₂, 0.1 mM TCEP, 0.05 mg/ml BSA, and 6% v/v glycerol) in 10 μl reactions. 0.1 nM (TATATA)₃ was mixed in as a non-binding loading control for quantification. Samples were electrophoresed on 4–20% TBE gels (Invitrogen) at 250 V for 30 min in cold 0.5x TB buffer, exposed to phosphor screens, and imaged with an Amersham Typhoon scanner (GE Life Sciences). Right, quantification of three independent experiments. Signal intensity was measured with ImageJ (NIH) and normalized to intensity of the loading control. Because the intensity of the bound species was lost due to trapping in the sample well, binding was quantified using depletion of the free probe. K_D,app values were calculated using the “One site – Specific binding” model in Prism 9 (GraphPad). Error bars represent standard error of the mean.