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Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

Abstract

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3′-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

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Figure 1: Structure of human pol θ polymerase domain.
Figure 2: DNA pol θ features insertions bearing conserved residues.
Figure 3
Figure 4: Primer extension assays and quantifications.
Figure 5: Primer extension assays using pol θ polymerase domain variants and single-stranded substrates.
Figure 6: The NCS two-fold axes in pol θ crystals, visualized and compared.

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Acknowledgements

These studies were funded by US National Institutes of Health grants R01 CA052040 (S.D.) and CA097175 (R.D.W.), grant RP130297 from the Cancer Prevention and Research Institute of Texas (R.D.W.) and the Grady F. Saunders PhD. Distinguished Research Professorship (R.D.W.). The GM/CA beamline at the Advanced Photon Source has been funded in whole or in part with federal funds from the US National Cancer Institute (ACB-12002) and the US National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the US DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

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Contributions

K.E.Z. performed crystallization, data collection and structure refinement. A.M.A. expressed and purified native and selenomethionine pol θ protein. P.A. obtained preliminary diffraction images at Diamond Light Source beamline I04. K.E.Z. expressed and purified mutant proteins and performed biochemical experiments. S.D. and R.D.W. oversaw the project. K.E.Z. wrote the manuscript with S.D. and R.D.W. All authors discussed the results and commented on the manuscript.

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Correspondence to Sylvie Doublié.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Pol θ helix O and incoming ddATP.

The cross-crystal NCS-averaged density-modified 2mFo-DFc electron density map (light blue mesh), calculated between THF–ddATP and dCMP–ddGTP data sets, is shown at a level of 1.3 σ in a wall-eyed stereo view about the active site, including the C-terminal region of helix-O, where a potential hydrogen bond is indicated (black dashes) between the hydroxyl group of Y2387 and the β-phosphate of the incoming ddATP.

Supplementary Figure 2 Pol θ active site and insert 2.

(a) The NCS averaged 2mFo-DFc electron density map (light blue mesh), calculated with regard to each of the four molecules of the ASU in the THF–ddATP structure, is contoured at 1.0 σ about insert 2 and the triphosphate tail of the incoming ddATP, and displayed in a wall-eyed stereo view. Interactions from conserved basic residues R2379 and K2383 are shown (black dashes). The putative salt bridge between R2254 of insert 2 (yellow) and the 3’-terminal phosphate of the primer strand stabilizes the primer terminus. (b) Thin amino acid side chains and the NCS averaged mFo-Dfc residual electron density map (red and green meshes), contoured at a level of ±3.5 σ, are added to the view appearing in panel a. The residual peak evident near the primer terminal 3’-OH suggests that an unmodeled metal ion might reside with partial occupancy in metal binding site A.

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Supplementary Figures 1 and 2 and Supplementary Tables 1 and 2 (PDF 581 kb)

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Zahn, K., Averill, A., Aller, P. et al. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair. Nat Struct Mol Biol 22, 304–311 (2015). https://doi.org/10.1038/nsmb.2993

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