Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures

Abstract

The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA–ssDNA and RecA–heteroduplex filaments. They show that ssDNA and ATP bind to RecA–RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP γ-phosphate is sensed across the RecA–RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson–Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

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Figure 1: Structure of the presynaptic nucleoprotein filament.
Figure 2: Each nucleotide triplet is bound by three consecutive RecA protomers.
Figure 3: The non-hydrolysable ATP analogue ADP-AlF 4 binds at a RecA–RecA interface.
Figure 4: Structure of the postsynaptic nucleoprotein filament.
Figure 5: Complementary-strand binding.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3CMW (RecA5–(dT)15), 3CMU (RecA6–(dT)18), 3CMX (RecA5–(dT)15–(dA)12), 3CMT (RecA5–d(T5C3AC2T4)–d(G2TG3)) and 3CMV (RecA4).

References

  1. 1

    Lusetti, S. L. & Cox, M. M. The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu. Rev. Biochem. 71, 71–100 (2002)

  2. 2

    Cromie, G. A., Connelly, J. C. & Leach, D. R. Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. Mol. Cell 8, 1163–1174 (2001)

  3. 3

    Khanna, K. K. & Jackson, S. P. DNA double-strand breaks: signaling, repair and the cancer connection. Nature Genet. 27, 247–254 (2001)

  4. 4

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

  5. 5

    Radding, C. M. Recombination activities of E. coli RecA protein. Cell 25, 3–4 (1981)

  6. 6

    Shinohara, A., Ogawa, H. & Ogawa, T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457–470 (1992)

  7. 7

    Seitz, E. M., Brockman, J. P., Sandler, S. J., Clark, A. J. & Kowalczykowski, S. C. RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. Genes Dev. 12, 1248–1253 (1998)

  8. 8

    Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998)

  9. 9

    Bell, C. E. Structure and mechanism of Escherichia coli RecA ATPase. Mol. Microbiol. 58, 358–366 (2005)

  10. 10

    McGrew, D. A. & Knight, K. L. Molecular design and functional organization of the RecA protein. Crit. Rev. Biochem. Mol. Biol. 38, 385–432 (2003)

  11. 11

    Cox, M. M. The bacterial RecA protein: Structure, function, and regulation. Top. Curr. Gen. 17, 53–94 (2007)

  12. 12

    Stasiak, A. & Di Capua, E. The helicity of DNA in complexes with RecA protein. Nature 299, 185–186 (1982)

  13. 13

    Yu, X., Jacobs, S. A., West, S. C., Ogawa, T. & Egelman, E. H. Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc. Natl Acad. Sci. USA 98, 8419–8424 (2001)

  14. 14

    Flory, J., Tsang, S. S. & Muniyappa, K. Isolation and visualization of active presynaptic filaments of RecA protein and single-stranded DNA. Proc. Natl Acad. Sci. USA 81, 7026–7030 (1984)

  15. 15

    Kelley De Zutter, J., Forget, A. L., Logan, K. M. & Knight, K. L. Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament. Structure 9, 47–55 (2001)

  16. 16

    Galletto, R., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006)

  17. 17

    Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006)

  18. 18

    VanLoock, M. S. et al. ATP-mediated conformational changes in the RecA filament. Structure 11, 187–196 (2003)

  19. 19

    DiCapua, E., Schnarr, M., Ruigrok, R. W., Lindner, P. & Timmins, P. A. Complexes of RecA protein in solution. A study by small angle neutron scattering. J. Mol. Biol. 214, 557–570 (1990)

  20. 20

    Morimatsu, K., Takahashi, M. & Norden, B. Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy. Proc. Natl Acad. Sci. USA 99, 11688–11693 (2002)

  21. 21

    Ogawa, T., Yu, X., Shinohara, A. & Egelman, E. H. Similarity of the yeast Rad51 filament to the bacterial RecA filament. Science 259, 1896–1899 (1993)

  22. 22

    Story, R. M., Weber, I. T. & Steitz, T. A. The structure of the E. coli RecA protein monomer and polymer. Nature 355, 318–325 (1992)

  23. 23

    Wu, Y., He, Y., Moya, I. A., Qian, X. & Luo, Y. Crystal structure of archaeal recombinase RadA: a snapshot of its extended conformation. Mol. Cell 15, 423–435 (2004)

  24. 24

    Conway, A. B. et al. Crystal structure of a Rad51 filament. Nature Struct. Mol. Biol. 11, 791–796 (2004)

  25. 25

    Yu, X., VanLoock, M. S., Yang, S., Reese, J. T. & Egelman, E. H. What is the structure of the RecA-DNA filament? Curr. Protein Pept. Sci. 5, 73–79 (2004)

  26. 26

    Stasiak, A., Egelman, E. H. & Howard-Flanders, P. Structure of helical RecA-DNA complexes. III. The structural polarity of RecA filaments and functional polarity in the RecA-mediated strand exchange reaction. J. Mol. Biol. 202, 659–662 (1988)

  27. 27

    Wang, Y. & Adzuma, K. Differential proximity probing of two DNA binding sites in the Escherichia coli RecA protein using photo-cross-linking methods. Biochemistry 35, 3563–3571 (1996)

  28. 28

    Malkov, V. A. & Camerini-Otero, R. D. Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops L1 (amino acid residues 157–164) and L2 (amino acid residues 195–209). J. Biol. Chem. 270, 30230–30233 (1995)

  29. 29

    Hortnagel, K. et al. Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair. J. Mol. Biol. 286, 1097–1106 (1999)

  30. 30

    Larminat, F., Cazaux, C., Germanier, M. & Defais, M. New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity. J. Bacteriol. 174, 6264–6269 (1992)

  31. 31

    Cox, J. M., Abbott, S. N., Chitteni-Pattu, S., Inman, R. B. & Cox, M. M. Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis. J. Biol. Chem. 281, 12968–12975 (2006)

  32. 32

    Nguyen, T. T., Muench, K. A. & Bryant, F. R. Inactivation of the RecA protein by mutation of histidine 97 or lysine 248 at the subunit interface. J. Biol. Chem. 268, 3107–3113 (1993)

  33. 33

    Morimatsu, K. & Horii, T. Analysis of the DNA binding site of Escherichia coli RecA protein. Adv. Biophys. 31, 23–48 (1995)

  34. 34

    Campbell, M. J. & Davis, R. W. Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation. J. Mol. Biol. 286, 417–435 (1999)

  35. 35

    Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997)

  36. 36

    Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R. & Wittinghofer, A. The GTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature 429, 197–201 (2004)

  37. 37

    Skiba, M. C. & Knight, K. L. Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli . J. Biol. Chem. 269, 3823–3828 (1994)

  38. 38

    Skiba, M. C., Logan, K. M. & Knight, K. L. Intersubunit proximity of residues in the RecA protein as shown by engineered disulfide cross-links. Biochemistry 38, 11933–11941 (1999)

  39. 39

    Kelley, J. A. & Knight, K. L. Allosteric regulation of RecA protein function is mediated by Gln194. J. Biol. Chem. 272, 25778–25782 (1997)

  40. 40

    Kunkel, T. A. & Bebenek, K. DNA replication fidelity. Annu. Rev. Biochem. 69, 497–529 (2000)

  41. 41

    Ishimori, K. et al. Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423. J. Mol. Biol. 264, 696–712 (1996)

  42. 42

    Mazin, A. V. & Kowalczykowski, S. C. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. EMBO J. 17, 1161–1168 (1998)

  43. 43

    Kurumizaka, H., Ikawa, S., Sarai, A. & Shibata, T. The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing. Arch. Biochem. Biophys. 365, 83–91 (1999)

  44. 44

    Leger, J. F., Robert, J., Bourdieu, L., Chatenay, D. & Marko, J. F. RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations. Proc. Natl Acad. Sci. USA 95, 12295–12299 (1998)

  45. 45

    Benedict, R. C. & Kowalczykowski, S. C. Increase of the DNA strand assimilation activity of RecA protein by removal of the C terminus and structure-function studies of the resulting protein fragment. J. Biol. Chem. 263, 15513–15520 (1988)

  46. 46

    Mikawa, T., Masui, R., Ogawa, T., Ogawa, H. & Kuramitsu, S. N-terminal 33 amino acid residues of Escherichia coli RecA protein contribute to its self-assembly. J. Mol. Biol. 250, 471–483 (1995)

  47. 47

    Dutreix, M., Burnett, B., Bailone, A., Radding, C. M. & Devoret, R. A partially deficient mutant, RecA1730, that fails to form normal nucleoprotein filaments. Mol. Gen. Genet. 232, 489–497 (1992)

  48. 48

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  49. 49

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  50. 50

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

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Acknowledgements

We thank D. King for mass spectroscopic analysis; H. Erdjument-Bromage for N-terminal sequencing; the staff of the Advanced Photon Source ID24 beamlines for help with data collection; M. Minto for administrative assistance; and the members of the Pavletich laboratory for help and discussions. This work was supported by the NIH and the Howard Hughes Medical Institute.

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Correspondence to Nikola P. Pavletich.

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The file contains Supplementary Tables 1-5, Supplementary Figures 1-11 with Legends and additional references (PDF 2798 kb)

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Chen, Z., Yang, H. & Pavletich, N. Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures. Nature 453, 489–494 (2008) doi:10.1038/nature06971

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