Structural basis of DNA polymerase θ mediated DNA end joining

Abstract DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.


Fig. S3 .
Fig. S3.Local cryo-EM density maps of complexes Ia and Ib.(a, b) Local cryo-EM maps for DNA in complexes Ia (a) and Ib (b).(c) Local cryo-EM maps for the Mg 2+ binding site in complex Ia.(d-h) Local cryo-EM maps for regions near the five insertion loops.

Fig. S4 .
Fig. S4.DNA interactions in A-family DNA polymerases.(a) Structure superimposition of the thumb domains in complex Ia and HsPol θ-pol bound with an RNA/DNA hybrid (6XBU).(b) Structure superimposition of the thumb domains in complex Ia and Taq polymerase (3KTQ).Key residues in the Taq thumb domain for DNA interactions are indicated.(c, d) Structures of the replicative A-family polymerases from bacteriophage T7 (c) and human mitochondria (d).The extended thumb loop (green) and the associated processivity factors (pale pink) are shown.

Fig. S5 .
Fig. S5.Active site features of high-and low-fidelity DNA polymerases.(a, b) Structure superimposition of the finger domains in complex Ia and HsPol θ-pol bound with an RNA/DNA hybrid (a) or with undamaged duplex DNA in the presence of an inhibitor (b).(c) Structure superimposition of the finger domains in complex Ia, Taq polymerase and Pol I suggests that complex Ia has a properly closed finger domain.(d) Structure superimposition of the active sites of complex Ia and Taq polymerase.(e) Comparison of the environment around the nascent base pair in Taq, Pol ν, and LcPol θ-pol (complex Ia).(f, g) Structure comparisons of Y-family Pol η incorporating against an undamaged base (cyan, 3MR2) and a CPD lesion (red, 3MR3) (f), and inserting a mismatched nucleotide (light green, 4J9K) (g).Key residues in the active sites are shown in sticks and labeled.

Fig. S6 .
Fig. S6.Cryo-EM data processing scheme of LcPol θ-pol complexed with the hairpin DNA.(a) A simplified flow chart of the cryo-EM data processing.(b) Local resolution of complex II.(c) Fourier Shell Correlation (FSC) curves for the overall resolution complex II.The golden standard (FSC=0.143) is used for resolution estimation.(d) Model-to-map fit between the full map and PDB coordinate of complex II.

Fig. S7 .
Fig. S7.Structural features of LcPol θ-pol complexed with the hairpin DNA.(a) Structure superimposition of the thumb domains in complexes Ia II.(b) Local cryo-EM map for the DNA binding site in complex II.(c) Finger domain movement in complexes Ia and II comparing to that in HsPol θ-pol bound with a dsDNA (4X0P) or an RNA/DNA hybrid (6XBU).(d) Local cryo-EM map for the active site.