Reassembly of shattered chromosomes in Deinococcus radiodurans

Abstract

Dehydration or desiccation is one of the most frequent and severe challenges to living cells1. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments2,3,4,5. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called ‘extended synthesis-dependent strand annealing’ (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become ‘sticky ends’ that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: DNA repair and synthesis after 7-kGy γ-irradiation of D. radiodurans.
Figure 2: Photolytic fragmentation of shattered D. radiodurans chromosomes reassembled in the presence of BrdU.
Figure 3: Single-cell detection of repair-associated newly synthesized single-stranded DNA by immunofluorescence microscopy.
Figure 4: Two-stage process of reconstitution of shattered chromosomes.

References

  1. 1

    Potts, M. Desiccation tolerance: a simple process? Trends Microbiol. 9, 553–559 (2001)

    CAS  Article  Google Scholar 

  2. 2

    Minton, K. W. DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans. Mol. Microbiol. 13, 9–15 (1994)

    CAS  Article  Google Scholar 

  3. 3

    Battista, J. R., Earl, A. M. & Park, M. J. Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol. 7, 362–365 (1999)

    CAS  Article  Google Scholar 

  4. 4

    Mattimore, V. & Battista, J. R. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. J. Bacteriol. 178, 633–637 (1996)

    CAS  Article  Google Scholar 

  5. 5

    Sanders, S. W. & Maxcy, R. B. Isolation of radiation-resistant bacteria without exposure to radiation. Appl. Environ. Microbiol. 38, 436–439 (1979)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Krasin, F. & Hutchinson, F. Repair of DNA double-strand breaks in Escherichia coli which requires recA function and the presence of duplicate genome. J. Mol. Biol. 116, 81–98 (1977)

    CAS  Article  Google Scholar 

  7. 7

    Fujimori, A. et al. Rad52 partially substitutes for Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells. EMBO J. 20, 5513–5520 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Dean, C. J., Feldschreiber, P. & Lett, J. T. Repair of X-ray damage to the deoxyribonucleic acid in Micrococcus radiodurans. Nature 209, 49–52 (1966)

    ADS  CAS  Article  Google Scholar 

  9. 9

    White, O. et al. Genome sequence of the radioresistant bacterium Deinococcus radiodurans. Science 286, 1571–1577 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Ferreira, A. C. et al. Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus. Extremophiles 3, 235–238 (1999)

    CAS  Article  Google Scholar 

  11. 11

    Narumi, I. Unlocking radiation resistance mechanisms: still a long way to go. Trends Microbiol. 11, 422–425 (2003)

    CAS  Article  Google Scholar 

  12. 12

    Cox, M. M. & Battista, J. R. Deinococcus radiodurans—the consummate survivor. Nature Rev. Microbiol. 3, 882–892 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Harsojo. Kitayama, S. & Matsuyama, A. Genome multiplicity and radiation resistance in Micrococcus radiodurans. J. Biochem. 90, 877–880 (1981)

    Article  Google Scholar 

  14. 14

    Levin-Zaidman, S. et al. Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science 299, 254–256 (2003)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Zimmerman, J. M. & Battista, J. R. A ring-like nucleoid is not necessary for radioresistance in Deinococcaceae. BMC Microbiol. 5, 17–27 (2005)

    Article  Google Scholar 

  16. 16

    Daly, M. J. & Minton, K. W. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans. J. Bacteriol. 178, 4461–4471 (1996)

    CAS  Article  Google Scholar 

  17. 17

    Meselson, M. & Stahl, F. W. The replication of DNA. Cold Spring Harb. Symp. Quant. Biol. 23, 9–12 (1958)

    CAS  Article  Google Scholar 

  18. 18

    Lion, M. B. Search for a mechanism for the increased sensitivity of 5-bromouracil-substituted DNA to ultraviolet radiation. II. Single-strand breaks in the DNA of irradiated 5-bromouracil-substituted T3 coliphage. Biochim. Biophys. Acta 209, 24–33 (1970)

    CAS  Article  Google Scholar 

  19. 19

    Lohman, P. H. M., Bootsma, D. & Hey, A. H. The influence of 5-bromodeoxyuridine on the induction of breaks in deoxyribonucleic acid of cultivated human cells by X-irradiation and ultraviolet light. Radiat. Res. 52, 627–641 (1972)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Lin, J. et al. Whole-genome shotgun optical mapping of Deinococcus radiodurans. Science 285, 1558–1562 (1999)

    CAS  Article  Google Scholar 

  21. 21

    Milligan, J. R. et al. DNA strand-break yield after incubation with base excision repair endonucleases implicate hydroxyl radical pairs in double-strand break formation. Int. J. Radiat. Biol. 76, 1475–1483 (2000)

    CAS  Article  Google Scholar 

  22. 22

    Lindahl, T. DNA repair enzymes. Annu. Rev. Biochem. 51, 61–87 (1982)

    CAS  Article  Google Scholar 

  23. 23

    Makarova, K. S. et al. Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol. Mol. Biol. Rev. 65, 44–79 (2001)

    CAS  Article  Google Scholar 

  24. 24

    Rayssiguier, C., Thaler, D. S. & Radman, M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342, 396–401 (1989)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Mennecier, S., Coste, G., Servant, P., Bailone, A. & Sommer, S. Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans. Mol. Genet. Genomics 272, 460–469 (2004)

    CAS  Article  Google Scholar 

  26. 26

    Rainey, F. A. et al. Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus from a single soil sample. Appl. Environ. Microbiol. 71, 5225–5235 (2005)

    CAS  Article  Google Scholar 

  27. 27

    Harris, D. R. et al. Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol. 2, 1629–1639 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Anderson, A. W., Nordon, H. C., Cain, R. F., Parrish, G. & Duggan, D. Studies on a radio-resistant micrococcus. I. Isolation, morphology, cultural characteristics and resistance to γ radiation. Food Technol. 10, 575–578 (1956)

    Google Scholar 

  29. 29

    Bonacossa de Almeida, C., Coste, G., Sommer, S. & Bailone, A. Quantification of RecA protein in Deinococcus radiodurans reveals involvement of RecA, but not LexA, in its regulation. Mol. Genet. Genomics 268, 28–41 (2002)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. Battista for initial help with Deinococcus and for the polA mutant; M. Meselson, B. Wagner, A. Stark, D. Zahradka and members of the M.R. lab for reading this manuscript and/or providing advice; M. Blazevic for technical assistance on γ-irradiation; and Mikula Radman for illustrations. K.Z. held a fellowship from the Necker Institute during work in M.R.'s laboratory. D.S. holds a Boehringer Ingelheim Foundation predoctoral fellowship and A.B.L. is a Marie Curie fellow. The laboratory in the Université de Paris-Descartes was funded by INSERM and the Necker Institute (Mixis/PLIVA contract); that in the Institut de Génétique et Microbiologie by EDF-France and CNRS (GEOMEX); that in the Institut Curie by EDF-France; and that in the Ruder Boskovic Institute by the Croatian Ministry of Science, Education and Sports. Author Contributions Experiments in Figs 1 and 2 and Supplementary Fig. 4 were carried out by K.Z.; those in Fig. 3 by D.S. A.B.L. provided expertise in microscopy; and the global experimental design was by M.R. The results of Fig. 1b, f, were obtained in, and with the scientific and material support of, the Institut de Génétique et Microbiologie; those of Fig. 1a, e, in the Institut Curie; those of refs Figs 1c, d, and Supplementary Fig. 4 in the Ruder Boskovic Institute, and those of Fig. 3 in the Université de Paris-Descartes.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Miroslav Radman.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Data

This file contains the full-length reviewed article. (DOC 1271 kb)

Supplementary Notes

This file contains Supplementary Methods, Supplementary Results, Supplementary Discussion and additional references. (PDF 93 kb)

Supplementary Figure 1

Analysis of repaired D. radiodurans DNA by 5-BrdU density labelling. (PPT 55 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zahradka, K., Slade, D., Bailone, A. et al. Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443, 569–573 (2006). https://doi.org/10.1038/nature05160

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing