Structural Basis for Polθ-Helicase DNA Binding and Microhomology-Mediated End-Joining

Abstract

DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.

Fig. 1.. Architecture of Polθ-hel-DNA complex.

(a) Schematic representation of the domain organization and construct design of Polθ-hel. WH, winged helix; HLH, helix-loop-helix. The regions either invisible in any of the cryoEM maps or excluded from the construct are colored in grey. (b) DNA substrate sequences used in this study. A 30-base pair DNA duplex with a poly(T) 3′-overhang ssDNA was prepared either with 6-nt MH sequence (CCCGGG) or without MH sequence. (c) Quantitation of DNA binding of Polθ-hel by rotational anisotropy. The FAM-labeled DNA with 3′-overhang ssDNA containing 9-nt poly(T) with MH, 11-nt poly(T) with MH, and 15-nt poly(T) without MH were used for the binding assay. (d) DNA gel shift assay with unlabeled DNA with 3′-overhang ssDNA containing 9-nt poly(T) with MH and 15-nt poly(T) without MH. (e) Orthogonal views of the cryoEM maps of Polθ-hel dimer in complex with 3′-overhangs with 9-nt poly(T) with MH (left), 15-nt poly(T) without MH (middle), and in apo form (right). (f) Orthogonal views of the atomic model of the Polθ-hel in MH annealed state. (g) Representative 2D class averages from the data sets of the complex with 15-nt poly(T) DNA without MH 3′-overhang DNA (left) and apo form (middle). The cryoEM map of the apo form tetramer is shown on the right.

Fig. 2.. Structural variability of the mobile domain D5.

(a) Cryo-EM density maps of Polθ-hel in the apo form, AMP-PNP-bound form, MH search state, and MH annealed states 1 and 2. The subdomain D5 (highlighted in blue) is bound to the helicase ring at the D2-D4 interface in the apo and AMP-PNP-bound forms but becomes invisible in the MH search state. In the MH annealed states, the D5 reemerges at the dimer interface in either one protomer (state 1) or both protomers (state 2). (b) Superimposition of a protomer from the apo form with the MH annealed state, highlighting the displacement of D5. The Cterminal end of the C-terminal helix is marked with an asterisk. (c) Positioning of the relocated D5 in the dimer context, depicted in a cylinder model (blue) against the rest of the Polθ-hel dimer shown in the surface representation (grey). An overlay of the cryoEM density over the atomic model of the relocated D5 is shown on the right. (d) Structure around the acidic patch of the U-shape structure in D5. Basic residues R637 and K640 of D4 in the other protomer electrostatically engage with the acidic patch. (e) Structure around the F839 of the U-shape structure in D5. The F839 is bound at the hydrophobic cavity formed by L614, F632, L769, and W771 of D4 in the other protomer. (f) Proposed model of the mobile D5 during the MMEJ.

Fig. 3.. DNA-helicase channel interactions.

(a) Overview of the 3′-overhang ssDNA (rendered as tube/slabs) threading through the helicase channel (surface model color-coded by subdomains). The direction of DNA translocation is indicated by an arrow. (b and c) Structure of ssDNA across the helicase channel, showing the atomic model of ssDNA (sticks) near the channel entrance (b) and exit (c), overlaid with the corresponding cryoEM density (mesh). (d) Detailed DNA-protein interactions around the channel entrance featuring the 3′-overhang DNA nucleotides T₁-T₄ (sticks) with surrounding amino acids (ribbons/sticks). (e) Detailed DNA-protein interactions around the channel exit featuring the 3′-overhang DNA nucleotides T₄-T₈ (sticks) with surrounding amino acids (ribbons/sticks). (f) Schematic of the ssDNA strand inside the Polθ-hel channel and interactions with residues from subdomains D1, D2, and D4. (g and h) Surface electrostatic potential of Polθ-hel. The positively charged patches near channel entrance (g) and exit (h) responsible for ssDNA capture. The surface area is colored according to the calculated electrostatic potential from −10.0 kT/e (red) to +10.0 kT/e (blue).

Fig. 4.. DNA-induced conformational changes in Polθ-hel dimer.

(a) Superimposition of the Polθ-hel dimer structure in the apo form (cylinder model in light pink) and the complexes with DNA in MH search state (light blue, left) and MH annealed state (lime, right). The bound DNA and the mobile domain D5 have been omitted from the models for clarity. (b) Superimposition of the Polθ-hel DNA complex in MH annealed state 1 dimer form with the dimer unit of the apo form tetramer. A steric clash between the DNA-bound dimer and the other dimer unit of the apo form tetramer is highlighted. (c) Superimposition of the apo form (ribbon model in light pink) and the MH annealed state (lime) near the exit of the helicase channel. The movement of the arginine helix in D1 is indicated by arrows. The ratchet helix was used for alignment in the superimposition.