Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination

Abstract

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2′s tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2′s function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2′s function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

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Figure 1: Purified PALB2 binds DNA.
Figure 2: PALB2 stimulates RAD51 strand exchange and DNA capture.
Figure 3: PALB2 interacts directly with RAD51.
Figure 4: PALB2 binds DNA with two distinct domains and stimulates RAD51-mediated D-loop formation.
Figure 5: A BRCA2 chimera protein (piBRCA2) recapitulates human BRCA2 properties.
Figure 6: A BRCA2 chimera stimulates RAD51-mediated D-loop formation and function with PALB2 in a synergistic manner to promote D-loop formation.
Figure 7: PALB2 is a homologous-recombination mediator protecting the RAD51 filament from disassembly.
Figure 8: Model of PALB2 and BRCA2 working synergistically to stimulate homologous recombination.

References

  1. 1

    Jemal, A. et al. Cancer statistics, 2009. CA-Cancer J. Clin. 59, 225–249 (2009).

    Google Scholar 

  2. 2

    Gudmundsdottir, K. & Ashworth, A. The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene 25, 5864–5874 (2006).

    Google Scholar 

  3. 3

    Venkitaraman, A.R. Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment. Annu. Rev. Pathol. 4, 461–487 (2009).

    Google Scholar 

  4. 4

    West, S.C. Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4, 435–445 (2003).

    Google Scholar 

  5. 5

    Davies, A.A. et al. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol. Cell 7, 273–282 (2001).

    Google Scholar 

  6. 6

    Esashi, F. et al. CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 434, 598–604 (2005).

    Google Scholar 

  7. 7

    Esashi, F., Galkin, V.E., Yu, X., Egelman, E.H. & West, S.C. Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat. Struct. Mol. Biol. 14, 468–474 (2007).

    Google Scholar 

  8. 8

    Carreira, A. et al. The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51. Cell 136, 1032–1043 (2009).

    Google Scholar 

  9. 9

    Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Google Scholar 

  10. 10

    Nimonkar, A.V., Ozsoy, A.Z., Genschel, J., Modrich, P. & Kowalczykowski, S.C. Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc. Natl. Acad. Sci. USA 105, 16906–16911 (2008).

    Google Scholar 

  11. 11

    Mimitou, E.P. & Symington, L.S. DNA end resection: many nucleases make light work. DNA Repair (Amst.) 8, 983–995 (2009).

    Google Scholar 

  12. 12

    Jazayeri, A. et al. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat. Cell Biol. 8, 37–45 (2006).

    Google Scholar 

  13. 13

    Yang, H., Li, Q., Fan, J., Holloman, W.K. & Pavletich, N.P. The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction. Nature 433, 653–657 (2005).

    Google Scholar 

  14. 14

    Xia, B. et al. Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2. Mol. Cell 22, 719–729 (2006).

    Google Scholar 

  15. 15

    Sy, S.M., Huen, M.S. & Chen, J. PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc. Natl. Acad. Sci. USA 106, 7155–7160 (2009).

    Google Scholar 

  16. 16

    Zhang, F. et al. PALB2 links BRCA1 and BRCA2 in the DNA-damage response. Curr. Biol. 19, 524–529 (2009).

    Google Scholar 

  17. 17

    Zhang, F., Fan, Q., Ren, K. & Andreassen, P.R. PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2. Mol. Cancer Res. 7, 1110–1118 (2009).

    Google Scholar 

  18. 18

    Rahman, N. et al. PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat. Genet. 39, 165–167 (2007).

    Google Scholar 

  19. 19

    Tischkowitz, M. et al. Analysis of PALB2/FANCN-associated breast cancer families. Proc. Natl. Acad. Sci. USA 104, 6788–6793 (2007).

    Google Scholar 

  20. 20

    Reid, S. et al. Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer. Nat. Genet. 39, 162–164 (2007).

    Google Scholar 

  21. 21

    Xia, B. et al. Fanconi anemia is associated with a defect in the BRCA2 partner PALB2. Nat. Genet. 39, 159–161 (2007).

    Google Scholar 

  22. 22

    Jones, S. et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 324, 217 (2009).

    Google Scholar 

  23. 23

    Ploquin, M. et al. Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases. Nucleic Acids Res. 35, 2719–2733 (2007).

    Google Scholar 

  24. 24

    McIlwraith, M.J. et al. Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins. J. Mol. Biol. 304, 151–164 (2000).

    Google Scholar 

  25. 25

    Rodrigue, A. et al. Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J. 25, 222–231 (2006).

    Google Scholar 

  26. 26

    Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine-139. J. Biol. Chem. 273, 5858–5868 (1998).

    Google Scholar 

  27. 27

    Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).

    Google Scholar 

  28. 28

    Celeste, A. et al. Genomic instability in mice lacking histone H2AX. Science 296, 922–927 (2002).

    Google Scholar 

  29. 29

    Sung, P. Mediating repair. Nat. Struct. Mol. Biol. 12, 213–214 (2005).

    Google Scholar 

  30. 30

    Saeki, H. et al. Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. Proc. Natl. Acad. Sci. USA 103, 8768–8773 (2006).

    Google Scholar 

  31. 31

    Bryant, H.E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).

    Google Scholar 

  32. 32

    Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).

    Google Scholar 

  33. 33

    Dray, E. et al. Enhancement of the RAD51 recombinase by the tumor suppressor PALB2. Nature Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1916 (26 September 2010).

  34. 34

    Sy, S.M., Huen, M.S., Zhu, Y. & Chen, J. PALB2 regulates recombinational repair through chromatin association and oligomerization. J. Biol. Chem. 284, 18302–18310 (2009).

    Google Scholar 

  35. 35

    Colavito, S. et al. Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption. Nucleic Acids Res. 37, 6754–6764 (2009).

    Google Scholar 

  36. 36

    Bugreev, D.V., Yu, X., Egelman, E.H. & Mazin, A.V. Novel pro- and anti-recombination activities of the Bloom's syndrome helicase. Genes Dev. 21, 3085–3094 (2007).

    Google Scholar 

  37. 37

    Hu, Y. et al. RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. Genes Dev. 21, 3073–3084 (2007).

    Google Scholar 

  38. 38

    Oliver, A.W., Swift, S., Lord, C.J., Ashworth, A. & Pearl, L.H. Structural basis for recruitment of BRCA2 by PALB2. EMBO Rep. 10, 990–996 (2009).

    Google Scholar 

  39. 39

    Mazloum, N., Zhou, Q. & Holloman, W.K. D-loop formation by Brh2 protein of Ustilago maydis. Proc. Natl. Acad. Sci. USA 105, 524–529 (2008).

    Google Scholar 

  40. 40

    Petalcorin, M.I., Sandall, J., Wigley, D.B. & Boulton, S.J. CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing. J. Mol. Biol. 361, 231–242 (2006).

    Google Scholar 

  41. 41

    Pellegrini, L. et al. Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 420, 287–293 (2002).

    Google Scholar 

  42. 42

    Haince, J.F., Rouleau, M., Hendzel, M.J., Masson, J.Y. & Poirier, G.G. Targeting poly(ADP-ribosyl)ation: a promising approach in cancer therapy. Trends Mol. Med. 11, 456–463 (2005).

    Google Scholar 

  43. 43

    Fong, P.C. et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N. Engl. J. Med. 361, 123–134 (2009).

    Google Scholar 

  44. 44

    Audeh, M.W. et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial. Lancet 376, 245–251 (2010).

    Google Scholar 

  45. 45

    Baumann, P., Benson, F.E., Hajibagheri, N. & West, S.C. Purification of human Rad51 protein by selective spermidine precipitation. Mutat. Res. DNA Repair 384, 65–72 (1997).

    Google Scholar 

  46. 46

    Henricksen, L.A., Umbricht, C.B. & Wold, M.S. Recombinant replication protein A: Expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994).

    Google Scholar 

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Acknowledgements

We thank E. Dray, P. Sung, S. Richard, I. Brodeur, E. Paquet, M. Tishkowitz and J. Vignard for helpful discussions, G. Hamel for technical help and I. Brodeur, J. Birraux and A. Rodrigue for critical reading of the manuscript. R.B. is supported by a doctoral scholarship from Le Fonds Québécois de la Recherche sur la Nature et les Technologies. This work was supported by funds from the Swiss National Science Foundation (grant 3103A-116275 to A.S.), the Cancer Institute of New Jersey (B.X.), the US National Cancer Institute (R01CA138804 to B.X.) and the Cancer Research Society (J.-Y.M.). B.X. is an American Cancer Society Research Scholar, and J.-Y.M. is a Fonds de la Recherche en Santé du Québec Senior II investigator.

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R.B., A.-M.D.-C., Y.C., H.L., H.C., A.S. and A.Z.S. conceived and carried out experiments. A.S. and B.X. contributed expertise. R.B. conceived the figures. J.-Y.M. provided guidance throughout and wrote the paper.

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Correspondence to Jean-Yves Masson.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods (PDF 4386 kb)

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Buisson, R., Dion-Côté, A., Coulombe, Y. et al. Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination. Nat Struct Mol Biol 17, 1247–1254 (2010). https://doi.org/10.1038/nsmb.1915

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