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Sustained active site rigidity during synthesis by human DNA polymerase μ

Nature Structural & Molecular Biology volume 21, pages 253–260 (2014)Cite this article

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Abstract

DNA polymerase μ (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3′ ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol μ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol μ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol μ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3′ ends.

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Figure 1: Structural features of the hPol μ Δ2 binary and precatalytic ternary complexes.
Figure 2: Structural characterization of nucleotide incorporation by hPol μ Δ2.
Figure 3: Electron density for hPol μ Δ2 structures.
Figure 4: Structural characteristics of the hPol μ apoprotein.
Figure 5: Biochemical characterization of wild type and loop 1 mutants of hPol μ.
Figure 6: Comparison of structural rigidity across the family-X polymerases.

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References

  1. Moon, A.F. et al. The X family portrait: structural insights into biological functions of X family polymerases. DNA Repair (Amst.) 6, 1709–1725 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  2. Nick McElhinny, S.A. & Ramsden, D.A. Sibling rivalry: competition between Pol X family members in V(d)J recombination and general double strand break repair. Immunol. Rev. 200, 156–164 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. DeRose, E.F. et al. Solution structure of polymerase μ's BRCT domain reveals an element essential for its role in nonhomologous end joining. Biochemistry 46, 12100–12110 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Mueller, G.A. et al. A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda. DNA Repair (Amst.) 7, 1340–1351 (2008).

    Article  CAS  Google Scholar 

  5. Bork, P. et al. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 11, 68–76 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Nick McElhinny, S.A. & Ramsden, D.A. Polymerase mu is a DNA-directed DNA/RNA polymerase. Mol. Cell. Biol. 23, 2309–2315 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ruiz, J.F. et al. Lack of sugar discrimination by human Pol μ requires a single glycine residue. Nucleic Acids Res. 31, 4441–4449 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Roettger, M.P., Fiala, K.A., Sompalli, S., Dong, Y. & Suo, Z. Pre-steady-state kinetic studies of the fidelity of human DNA polymerase μ. Biochemistry 43, 13827–13838 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Bertocci, B., De Smet, A., Berek, C., Weill, J.C. & Reynaud, C.A. Immunoglobulin κ light chain gene rearrangement is impaired in mice deficient for DNA polymerase mu. Immunity 19, 203–211 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Bertocci, B., De Smet, A., Weill, J.C. & Reynaud, C.A. Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(d)J recombination in vivo. Immunity 25, 31–41 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Gozalbo-López, B. et al. A role for DNA polymerase μ in the emerging DJH rearrangements of the postgastrulation mouse embryo. Mol. Cell. Biol. 29, 1266–1275 (2009).

    Article  PubMed  Google Scholar 

  12. Lucas, D. et al. Altered hematopoiesis in mice lacking DNA polymerase μ is due to inefficient double-strand break repair. PLoS Genet. 5, e1000389 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Chayot, R., Danckaert, A., Montagne, B. & Ricchetti, M. Lack of DNA polymerase μ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence. DNA Repair (Amst.) 9, 1187–1199 (2010).

    Article  CAS  Google Scholar 

  14. Nick McElhinny, S.A. et al. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol. Cell 19, 357–366 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Andrade, P., Martin, M.J., Juarez, R., Lopez de Saro, F. & Blanco, L. Limited terminal transferase in human DNA polymerase μ defines the required balance between accuracy and efficiency in NHEJ. Proc. Natl. Acad. Sci. USA 106, 16203–16208 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Domínguez, O. et al. DNA polymerase mu (Pol μ), homologous to TdT, could act as a DNA mutator in eukaryotic cells. EMBO J. 19, 1731–1742 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Juárez, R., Ruiz, J.F., Nick McElhinny, S.A., Ramsden, D. & Blanco, L. A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis. Nucleic Acids Res. 34, 4572–4582 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Romain, F., Barbosa, I., Gouge, J., Rougeon, F. & Delarue, M. Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region. Nucleic Acids Res. 37, 4642–4656 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Martin, M.J., Juarez, R. & Blanco, L. DNA-binding determinants promoting NHEJ by human Polμ. Nucleic Acids Res. 40, 11389–11403 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Moon, A.F. et al. Structural insight into the substrate specificity of DNA Polymerase μ. Nat. Struct. Mol. Biol. 14, 45–53 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Batra, V.K. et al. Magnesium-induced assembly of a complete DNA polymerase catalytic complex. Structure 14, 757–766 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Pedersen, L.C. & Kunkel, T.A. Role of the catalytic metal during polymerization by DNA polymerase lambda. DNA Repair (Amst.) 6, 1333–1340 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  23. Batra, V.K., Beard, W.A., Shock, D.D., Pedersen, L.C. & Wilson, S.H. Structures of DNA polymerase β with active-site mismatches suggest a transient abasic site intermediate during misincorporation. Mol. Cell 30, 315–324 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bebenek, K., Pedersen, L.C. & Kunkel, T.A. Replication infidelity via a mismatch with Watson-Crick geometry. Proc. Natl. Acad. Sci. USA 108, 1862–1867 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cavanaugh, N.A. et al. Molecular insights into DNA polymerase deterrents for ribonucleotide insertion. J. Biol. Chem. 286, 31650–31660 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Pedersen, L.C. & Kunkel, T.A. Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase. Cell 124, 331–342 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Gosavi, R.A., Moon, A.F., Kunkel, T.A., Pedersen, L.C. & Bebenek, K. The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ. Nucleic Acids Res. 40, 7518–7527 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Picher, A.J. et al. Promiscuous mismatch extension by human DNA polymerase lambda. Nucleic Acids Res. 34, 3259–3266 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Franklin, M.C., Wang, J. & Steitz, T.A. Structure of the replicating complex of a pol α family DNA polymerase. Cell 105, 657–667 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Rodriguez, A.C., Park, H.W., Mao, C. & Beese, L.S. Crystal structure of a pol α family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9° N-7. J. Mol. Biol. 299, 447–462 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Wing, R.A., Bailey, S. & Steitz, T.A. Insights into the replisome from the structure of a ternary complex of the DNA polymerase III α-subunit. J. Mol. Biol. 382, 859–869 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wu, E.Y. & Beese, L.S. The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an “ajar” intermediate conformation in the nucleotide selection mechanism. J. Biol. Chem. 286, 19758–19767 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Kunkel, T.A. & Pedersen, L.C. A closed conformation for the Pol λ catalytic cycle. Nat. Struct. Mol. Biol. 12, 97–98 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Doherty, A.J., Serpell, L.C. & Ponting, C.P. The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res. 24, 2488–2497 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Davis, B.J., Havener, J.M. & Ramsden, D.A. End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining. Nucleic Acids Res. 36, 3085–3094 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bebenek, K., Garcia-Diaz, M., Zhou, R.Z., Povirk, L.F. & Kunkel, T.A. Loop 1 modulates the fidelity of DNA polymerase λ. Nucleic Acids Res. 38, 5419–5431 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Radhakrishnan, R. et al. Regulation of DNA repair fidelity by molecular checkpoints: “gates” in DNA polymerase β's substrate selection. Biochemistry 45, 15142–15156 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Yang, L., Beard, W.A., Wilson, S.H., Broyde, S. & Schlick, T. Polymerase β simulations suggest that Arg258 rotation is a slow step rather than large subdomain motions per se. J. Mol. Biol. 317, 651–671 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Li, Y. & Schlick, T. Modeling DNA polymerase μ motions: subtle transitions before chemistry. Biophys. J. 99, 3463–3472 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Valley, C.C. et al. The methionine-aromatic motif plays a unique role in stabilizing protein structure. J. Biol. Chem. 287, 34979–34991 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Delarue, M. et al. Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. EMBO J. 21, 427–439 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gouge, J., Rosario, S., Romain, F., Beguin, P. & Delarue, M. Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism. J. Mol. Biol. 425, 4334–4352 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Bollum, F.J. Thermal conversion of nonpriming deoxyribonucleic acid to primer. J. Biol. Chem. 234, 2733–2734 (1959).

    CAS  PubMed  Google Scholar 

  44. Kato, K.I., Goncalves, J.M., Houts, G.E. & Bollum, F.J. Deoxynucleotide-polymerizing enzymes of calf thymus gland: II. Properties of the terminal deoxynucleotidyltransferase. J. Biol. Chem. 242, 2780–2789 (1967).

    CAS  PubMed  Google Scholar 

  45. Biertümpfel, C. et al. Structure and mechanism of human DNA polymerase η. Nature 465, 1044–1048 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Ummat, A. et al. Human DNA polymerase η is pre-aligned for dNTP binding and catalysis. J. Mol. Biol. 415, 627–634 (2012).

    Article  CAS  PubMed  Google Scholar 

  47. Beard, W.A., Shock, D.D., Batra, V.K., Pedersen, L.C. & Wilson, S.H. DNA polymerase β substrate specificity: side chain modulation of the “A-rule”. J. Biol. Chem. 284, 31680–31689 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Moon, A.F., Mueller, G.A., Zhong, X. & Pedersen, L.C. A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction. Protein Sci. 19, 901–913 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Chayen, N.E. Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques. Acta Crystallogr. D Biol. Crystallogr. 54, 8–15 (1998).

    Article  CAS  PubMed  Google Scholar 

  50. Zwart, P.H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  54. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lovell, S.C. et al. Structure validation by Cα geometry: φ, ψψψ and Cβ deviation. Proteins 50, 437–450 (2003).

    Article  CAS  PubMed  Google Scholar 

  56. Nick McElhinny, S.A., Snowden, C.M., McCarville, J. & Ramsden, D.A. Ku recruits the XRCC4-ligase IV complex to DNA ends. Mol. Cell. Biol. 20, 2996–3003 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Prasad, R., Kumar, A., Widen, S.G., Casas-Finet, J.R. & Wilson, S.H. Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase β by UV cross-linking. J. Biol. Chem. 268, 22746–22755 (1993).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank G. Mueller and B. Beard for critical reading of the manuscript. This research was supported by the Division of Intramural Research of the US National Institute of Environmental Health Sciences, National Institutes of Health (NIH) grants ES102645 (L.C.P.), and ES065070 (T.A.K.) and by NIH grant CA097096 (D.A.R.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences contract W-31-109-Eng-38.

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Authors and Affiliations

  1. Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Andrea F Moon, Thomas A Kunkel, Katarzyna Bebenek & Lars C Pedersen

  2. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    John M Pryor & Dale A Ramsden

  3. Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Thomas A Kunkel & Katarzyna Bebenek

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  1. Andrea F Moon
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Contributions

A.F.M., L.C.P., K.B., D.A.R. and T.A.K. designed research; A.F.M., K.B. and J.M.P. performed research; and all authors contributed to data analysis and manuscript preparation.

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Correspondence to Katarzyna Bebenek.

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Integrated supplementary information

Supplementary Figure 1 Comparison of wild-type full-length and catalytic-domain (Pro138–Ala494) constructs of hPol μ in single-nucleotide gap-filling.

A steady-state polymerase activity assay was performed in order to compare wildtype full-length or truncated Pol μ constructs (5nM), on a single-nucleotide gapped DNA substrate (no enzyme control, C). The percentage of primer extension was calculated for each construct, at two different time points. The two constructs behaved indistinguishably in this assay.

Supplementary Figure 2 Protein engineering of hPol μ Δ2.

(a) Comparison of the X-ray crystal structures of mouse Pol μ (PDB ID code 2IHM1, purple) and mouse TdT (PDB ID code 1JMS2, orange). The relative positions of Loop 1 (green, from mTdT) and Loop 2 (red, from mPol μ) are displayed. (b) Ribbon diagram showing the ordered and disordered regions of Loop 2 in mTdT (orange) and mPol μ (red). (c) ClustalW3 sequence alignments of Loop1 and Loop 2 in mammalian orthologs of Pol μ. Loop1 is boxed in green. Regions of decreased sequence conservation in Loop2 are boxed in red, and were subsequently deleted by site-directed mutagenesis. His363 (magenta), Met382 (green), and Phe385 (cyan) are clearly marked. (d) ClustalW sequence alignments of Loop 2 in mammalian orthologs of TdT. Loop1 is boxed in green, and regions that are structurally homologous to the deleted region of Loop 2 in hPol μ are boxed in red. Phe401 is marked in cyan. (e) Structure of mPol μ (purple), displaying the location of Loop 2 (red), distal from the active site (marked by the incoming nucleotide, cyan), and the DNA binding cleft (khaki ribbon and surface rendering). (f) Structure of engineered Loop 2 from hPol μ Δ2 binary complex (blue), compared to mPol μ (purple). Residues Pro398-Pro410 were deleted, and β-strands 4 and 5 fused by addition of a glycine residue (labeled Gly410 and marked by an asterisk). All structural figures were generated using PyMOL4.

Supplementary Figure 3 Activity assays with wild-type hPol μ and the hPol μ Δ2 variant.

(a) Comparison of wildtype and Δ2 variant hPol μ template-dependent synthesis activity on a single nucleotide gapped DNA substrate. (b) Comparison of wildtype and Δ2 hPol μ template-independent synthesis activity on a single-stranded oligo-dT DNA substrate. (c) Comparison of wildtype and hPol μ Δ2 during in vitro NHEJ assays. Top, schematic diagram illustrating the structure of the DNA substrates used in the NHEJ assay. Bottom, ligation products of NHEJ synthesized by either wildtype or hPol μ Δ2.

Supplementary Figure 4 Implications of loop 1 flexibility for substrate stabilization during DSB repair by NHEJ.

(a) Model of a noncomplementary DSB substrate (purple) bound to the protein component of the hPol μ Δ2 pre-catalytic ternary complex (orange). The primer (strand P) terminus (blue) is unpaired, opposite the discontinuity in the template strand (strand T). The incoming nucleotide (cyan) is correctly paired opposite the templating base (magenta). Location of the disordered Loop1 is marked in green. (b) Hypothetical Loop1 conformations were manually generated for the single-nucleotide gapped ternary complex (green) and for a noncomplementary DSB substrate (red).

Supplementary Figure 5 Biochemical characterization of wild-type and loop 1 mutants of hPol μ.

(a) Uncropped gel from Figure 5b. (b) Uncropped gel from Figure 5c (top panel). (c) Uncropped gel from Figure 5c (bottom panel). All wells in (b) and (c) also contain human Ku (10 nM) and XRCC4/Ligase IV complex (20 nM) in addition to the indicated Family X polymerase (0.5 nM).

Supplementary Figure 6 The multiple cloning site of the pGEXM expression vector.

The vestigial thrombin protease cleavage site from the parental pGEX-4T3 expression vector is highlighted in blue. The Tobacco Etch Virus (TEV) protease cleavage site is shown in green, with a green line clearly delineating the actual site of cleavage. The BamHI restriction site between the two regions encoding the thrombin and TEV protease sites has been deactivated by site directed mutagenesis (blue asterisk), a silent mutation which does not affect the protein sequence. The multicloning site from the pMALX expression vector is shown5, with the pertinent restriction sites marked. Three TGA stop codons (red) are situated immediately downstream of the multicloning site, one in each reading frame. Locations of sequencing primers 5'pGEX and 3'pGEX are shown with directional arrows.

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Moon, A., Pryor, J., Ramsden, D. et al. Sustained active site rigidity during synthesis by human DNA polymerase μ. Nat Struct Mol Biol 21, 253–260 (2014). https://doi.org/10.1038/nsmb.2766

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