Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Homology-driven chromatin remodeling by human RAD54

Nature Structural & Molecular Biology volume 14, pages 397–405 (2007)Cite this article

Abstract

Human RAD51 and RAD54 are key players in homologous recombination, a process that requires homology recognition and strand invasion by a RAD51–single-stranded DNA (ssDNA) nucleoprotein filament and chromatin remodeling by RAD54. Here we use in vitro chromatin reconstitution systems to show that RAD51-ssDNA stimulates RAD54-dependent chromatin remodeling in a homology-dependent, polarity-independent manner. This stimulation was not seen with RAD54B or other remodelers. Chromatin remodeling by RAD54 enabled strand invasion by RAD51-ssDNA on nucleosomal templates, which was homology- and polarity-dependent. Three natural RAD54 mutants found in primary cancer cells showed specific defects in remodeling or in the RAD54-RAD51 interaction. We propose that RAD54 is recruited by RAD51-ssDNA filament to the chromatin of the intact chromosome and that it remodels that chromatin to facilitate accessibility for strand exchange.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: ATP-dependent chromatin remodeling by RAD54 and RAD54B.
Figure 2: RAD54 remodeling activity was stimulated by RAD51-ssDNA filaments assembled using ssDNA identical to different regions of template DNA.
Figure 3: RAD51-ssDNA filaments enhance the accessibility of HhaI sites on the G5E4 array catalyzed by RAD54 in a homology-dependent manner.
Figure 4: RAD54 facilitates strand invasion on chromatin by RAD51-ssDNA filament.
Figure 5: Activities of RAD54 mutants.
Figure 6: Two alternative models for homology search on chromatin.

Similar content being viewed by others

References

  1. Neale, M.J. & Keeney, S. Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158 (2006).

    Article  CAS  Google Scholar 

  2. Michel, B. et al. Rescue of arrested replication forks by homologous recombination. Proc. Natl. Acad. Sci. USA 98, 8181–8188 (2001).

    Article  CAS  Google Scholar 

  3. Tarsounas, M. & West, S.C. Recombination at mammalian telomeres: an alternative mechanism for telomere protection and elongation. Cell Cycle 4, 672–674 (2005).

    Article  CAS  Google Scholar 

  4. Couedel, C. et al. Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev. 18, 1293–1304 (2004).

    Article  CAS  Google Scholar 

  5. Mills, K.D. et al. Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability. Genes Dev. 18, 1283–1292 (2004).

    Article  CAS  Google Scholar 

  6. Baumann, P. & West, S.C. Role of the human RAD51 protein in homologous recombination and double- stranded-break repair. Trends Biochem. Sci. 23, 247–251 (1998).

    Article  CAS  Google Scholar 

  7. Heyer, W.D., Li, X., Rolfsmeier, M. & Zhang, X.P. Rad54: the Swiss Army knife of homologous recombination? Nucleic Acids Res. 34, 4115–4125 (2006).

    Article  CAS  Google Scholar 

  8. Tan, T.L., Kanaar, R. & Wyman, C. Rad54, a Jack of all trades in homologous recombination. DNA Repair (Amst.) 2, 787–794 (2003).

    Article  Google Scholar 

  9. Van Komen, S., Petukhova, G., Sigurdsson, S., Stratton, S. & Sung, P. Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol. Cell 6, 563–572 (2000).

    Article  CAS  Google Scholar 

  10. Mazin, A.V., Alexeev, A.A. & Kowalczykowski, S.C. A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J. Biol. Chem. 278, 14029–14036 (2003).

    Article  CAS  Google Scholar 

  11. Solinger, J.A., Kiianitsa, K. & Heyer, W.D. Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol. Cell 10, 1175–1188 (2002).

    Article  CAS  Google Scholar 

  12. Bugreev, D.V., Mazina, O.M. & Mazin, A.V. Rad54 protein promotes branch migration of Holliday junctions. Nature 442, 590–593 (2006).

    Article  CAS  Google Scholar 

  13. Alexeev, A., Mazin, A. & Kowalczykowski, S.C. Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. Nat. Struct. Biol. 10, 182–186 (2003).

    Article  CAS  Google Scholar 

  14. Alexiadis, V., Lusser, A. & Kadonaga, J.T. A conserved N-terminal motif in Rad54 is important for chromatin remodeling and homologous strand pairing. J. Biol. Chem. 279, 27824–27829 (2004).

    Article  CAS  Google Scholar 

  15. Wolner, B. & Peterson, C.L. ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break. J. Biol. Chem. 280, 10855–10860 (2005).

    Article  CAS  Google Scholar 

  16. Jaskelioff, M., Van Komen, S., Krebs, J.E., Sung, P. & Peterson, C.L. Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J. Biol. Chem. 278, 9212–9218 (2003).

    Article  CAS  Google Scholar 

  17. Wesoly, J. et al. Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis. Mol. Cell. Biol. 26, 976–989 (2006).

    Article  CAS  Google Scholar 

  18. Mazin, A.V., Bornarth, C.J., Solinger, J.A., Heyer, W.D. & Kowalczykowski, S.C. Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol. Cell 6, 583–592 (2000).

    Article  CAS  Google Scholar 

  19. Narlikar, G.J., Phelan, M.L. & Kingston, R.E. Generation and interconversion of multiple distinct nucleosomal states as a mechanism for catalyzing chromatin fluidity. Mol. Cell 8, 1219–1230 (2001).

    Article  CAS  Google Scholar 

  20. Logie, C. & Peterson, C.L. Catalytic activity of the yeast SWI/SNF complex on reconstituted nucleosome arrays. EMBO J. 16, 6772–6782 (1997).

    Article  CAS  Google Scholar 

  21. Polach, K.J. & Widom, J. Restriction enzymes as probes of nucleosome stability and dynamics. Methods Enzymol. 304, 278–298 (1999).

    Article  CAS  Google Scholar 

  22. Hamiche, A., Sandaltzopoulos, R., Gdula, D.A. & Wu, C. ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF. Cell 97, 833–842 (1999).

    Article  CAS  Google Scholar 

  23. Langst, G. & Becker, P.B. ISWI induces nucleosome sliding on nicked DNA. Mol. Cell 8, 1085–1092 (2001).

    Article  CAS  Google Scholar 

  24. Fan, H.Y., He, X., Kingston, R.E. & Narlikar, G.J. Distinct strategies to make nucleosomal DNA accessible. Mol. Cell 11, 1311–1322 (2003).

    Article  CAS  Google Scholar 

  25. Mazina, O.M. & Mazin, A.V. Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J. Biol. Chem. 279, 52042–52051 (2004).

    Article  CAS  Google Scholar 

  26. Sung, P. & Robberson, D.L. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453–461 (1995).

    Article  CAS  Google Scholar 

  27. Matsuda, M. et al. Mutations in the RAD54 recombination gene in primary cancers. Oncogene 18, 3427–3430 (1999).

    Article  CAS  Google Scholar 

  28. Smirnova, M., Van Komen, S., Sung, P. & Klein, H.L. Effects of tumor-associated mutations on Rad54 functions. J. Biol. Chem. 279, 24081–24088 (2004).

    Article  CAS  Google Scholar 

  29. Hsieh, P., Camerini-Otero, C.S. & Camerini-Otero, R.D. The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA. Proc. Natl. Acad. Sci. USA 89, 6492–6496 (1992).

    Article  CAS  Google Scholar 

  30. Alexiadis, V. & Kadonaga, J.T. Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16, 2767–2771 (2002).

    Article  CAS  Google Scholar 

  31. Sigurdsson, S., Van Komen, S., Petukhova, G. & Sung, P. Homologous DNA pairing by human recombination factors Rad51 and Rad54. J. Biol. Chem. 277, 42790–42794 (2002).

    Article  CAS  Google Scholar 

  32. Thoma, N.H. et al. Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat. Struct. Mol. Biol. 12, 350–356 (2005).

    Article  Google Scholar 

  33. Golub, E.I., Gupta, R.C., Haaf, T., Wold, M.S. & Radding, C.M. Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA. Nucleic Acids Res. 26, 5388–5393 (1998).

    Article  CAS  Google Scholar 

  34. Raschle, M., Van Komen, S., Chi, P., Ellenberger, T. & Sung, P. Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54. J. Biol. Chem. 279, 51973–51980 (2004).

    Article  CAS  Google Scholar 

  35. Folta-Stogniew, E., O'Malley, S., Gupta, R., Anderson, K.S. & Radding, C.M. Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein. Mol. Cell 15, 965–975 (2004).

    Article  CAS  Google Scholar 

  36. Gupta, R.C., Folta-Stogniew, E., O'Malley, S., Takahashi, M. & Radding, C.M. Rapid exchange of A:T base pairs is essential for recognition of DNA homology by human Rad51 recombination protein. Mol. Cell 4, 705–714 (1999).

    Article  CAS  Google Scholar 

  37. Voloshin, O.N. & Camerini-Otero, R.D. Synaptic complex revisited; a homologous recombinase flips and switches bases. Mol. Cell 15, 846–847 (2004).

    Article  CAS  Google Scholar 

  38. Swagemakers, S.M., Essers, J., de Wit, J., Hoeijmakers, J.H. & Kanaar, R. The human RAD54 recombinational DNA repair protein is a double-stranded DNA-dependent ATPase. J. Biol. Chem. 273, 28292–28297 (1998).

    Article  CAS  Google Scholar 

  39. Tanaka, K., Kagawa, W., Kinebuchi, T., Kurumizaka, H. & Miyagawa, K. Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54. Nucleic Acids Res. 30, 1346–1353 (2002).

    Article  CAS  Google Scholar 

  40. Yoon, D., Wang, Y., Stapleford, K., Wiesmuller, L. & Chen, J. P53 inhibits strand exchange and replication fork regression promoted by human Rad51. J. Mol. Biol. 336, 639–654 (2004).

    Article  CAS  Google Scholar 

  41. Aalfs, J.D., Narlikar, G.J. & Kingston, R.E. Functional differences between the human ATP-dependent nucleosome remodeling proteins BRG1 and SNF2H. J. Biol. Chem. 276, 34270–34278 (2001).

    Article  CAS  Google Scholar 

  42. Owen-Hughes, T. & Workman, J.L. Remodeling the chromatin structure of a nucleosome array by transcription factor-targeted trans-displacement of histones. EMBO J. 15, 4702–4712 (1996).

    Article  CAS  Google Scholar 

  43. Shrader, T.E. & Crothers, D.M. Artificial nucleosome positioning sequences. Proc. Natl. Acad. Sci. USA 86, 7418–7422 (1989).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Kanaar (Erasmus MC), J. Chen (University of Delaware) and K. Miyagawa (Hiroshima University) for plasmids pFASTBac1/6His-HHR54-HA, pTrcHisB/hRAD51 and pFASTBacHTc/6His-RAD54B, respectively, the members of the Kingston laboratory for critical comments and J. Garlick for help with the Sf9 cell culture. This work was supported by US National Institutes of Health, National Cancer Institute grant CA-093660 (H.-Y.F) and US National Institutes of Health grant GM48405 (R.E.K.).

Author information

Author notes

  1. Hua-Ying Fan

    Present address: Present address: Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, W470, Philadelphia, Pennsylvania 19111, USA.,

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, 02114, Massachusetts, USA

    Zhaoqing Zhang, Hua-Ying Fan, Joseph A Goldman & Robert E Kingston

Authors
  1. Zhaoqing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hua-Ying Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph A Goldman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert E Kingston
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.Z. designed and executed the experiments. H.-Y.F. established and optimized the mononucleosome-related experiments. J.A.G. optimized the REA reactions on nucleosomal arrays. Z.Z. and R.E.K. interpreted the data and prepared the manuscript.

Corresponding author

Correspondence to Robert E Kingston.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Recombinant proteins purified from Sf9 cells. (PDF 110 kb)

Supplementary Fig. 2

Control reactions for the remodeling activity of RAD54 and RAD54B. (PDF 276 kb)

Supplementary Fig. 3

RAD51-ssDNA enhances the chromatin-remodeling activity of RAD54 specifically. (PDF 101 kb)

Supplementary Fig. 4

Stoichiometry of RAD51-ssDNA stimulation to the remodeling of the G5E4 array by RAD54. (PDF 132 kb)

Supplementary Fig. 5

Nucleosome occupancy on the G5E4 circular chromatin. (PDF 157 kb)

Supplementary Fig. 6

The effects of different chromatin-remodeling enzymes on strand invasion by RAD51-ssDNA. (PDF 122 kb)

Supplementary Table 1

Vmax of ATP hydrolysis and Km for different substrates, derived from ATPase assays. (PDF 54 kb)

Supplementary Table 2

Sequences of oligonucleotides used for RAD51-ssDNA filament assembly. (PDF 50 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, Z., Fan, HY., Goldman, J. et al. Homology-driven chromatin remodeling by human RAD54. Nat Struct Mol Biol 14, 397–405 (2007). https://doi.org/10.1038/nsmb1223

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1223

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing