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Ringlike Structure of the Deinococcus radiodurans Genome: A Key to Radioresistance?

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Science  10 Jan 2003:
Vol. 299, Issue 5604, pp. 254-256
DOI: 10.1126/science.1077865

Abstract

The bacterium Deinococcus radiodurans survives ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms. How D. radioduransaccurately reconstructs its genome from hundreds of radiation-generated fragments in the absence of an intact template is unknown. Here we show that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. We propose that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroids, radiation-generated free DNA ends are held together, which may facilitate template-independent yet error-free joining of DNA breaks.

Deinococcus radiodurans is capable of surviving 15,000 grays (Gy) of ionizing radiation, whereas doses below 10 Gy are lethal to all other organisms (1, 2). The bacterium's phenomenal radioresistance derives from its ability to accurately mend hundreds of double-strand DNA breaks (2–7). This mending is unlikely to occur by homologous recombination, the only known mechanism for high-fidelity repair of double-strand breaks, because this mechanism is ineffective when chromosomes are extensively shattered. The enigmatic nature of D. radiodurans' radioresistance is highlighted by the finding that this organism encodes a typical bacterial complement of DNA repair enzymes (7–9).

To investigate the factors responsible for the radioresistance ofD. radiodurans, we studied the morphology of the bacterium. Our scanning electron microscopy analysis (10) confirmed previous observations (11) that each D. radiodurans cell has two perpendicular furrows that result in a tetrad morphology. This morphology is exhibited by all cells in a stationary state and by >90% of actively growing cells (fig. S1A). Differential interference contrast and fluorescence microscopy (fig. S1, B to D), as well as integrated fluorescence intensity measurements (10), revealed that in stationary-state bacteria, the four compartments contain an equal amount of DNA. Uniform compartmentalization of DNA was also detected in stationary-state cells that had been subjected to ultraviolet (UV) or ionizing radiation and then allowed to recover in fresh growth medium for 1 hour. Because starved D. radiodurans bacteria carry four genome copies (7), it follows that each compartment contains a single genome. These findings imply that the extensive DNA repair that occurs in growing as well as in stationary-state (12)D. radiodurans cells within the first hour after irradiation (13) cannot involve homologous recombination, because no template is available.

Transmission electron microscopy studies (10,11) showed that a membranous framework demarcates the four compartments of a single D. radiodurans cell, but orifices were evident in the internal membranes of both growing and stationary-state cells (Fig. 1). Chromatin was present in all compartments, and it adopted a distinctive toroidal shape (Fig. 1, A and B). DNA labeling (10) revealed that, whereas DNA toroids are present in all compartments of stationary-state bacteria, the nucleoid in one or two compartments of growing cells is dispersed (Fig. 1, C and D). This dispersed DNA morphology, which is common in vegetative bacteria, accounts for metabolic activity because it allows access for enzymes that mediate replication and transcription (14).

Figure 1

Transmission electron micrographs of D. radiodurans cells that are (A) actively growing (8 hours old) or (B) in a stationary state (6 days old). The densely stained particles are ribosomes; ribosome-free spaces contain chromatin (14). Statistical analysis (10) indicates that all stationary cells have a tetrad morphology, although this morphology was not detectable in all thin sections. Orifices within internal membranes were discerned in all specimens. (C) Actively growing cells stained with the DNA-specific reagent osmium-ammine-SO2 (10), which produces darkly stained chromatin. DNA toroids are present in three compartments, whereas the chromatin in the upper right compartment exhibits a dispersed morphology. (D) DNA staining of 6-day-old stationary-state cells. Identical morphology is displayed by cells incubated for 60 min after exposure to ionizing radiation (15 kGy). (E) DNA staining of 6-day-old cells incubated for 90 min after exposure to 15 kGy of ionizing radiation, showing disruption of DNA toroids. (F) DNA staining of 6-day-old cells incubated for 3 hours after exposure to ionizing radiation (15 kGy), showing DNA spreading between two compartments through a membranous orifice and subsequent nucleoid fusion. Scale bars, 400 nm.

We next examined the morphology of D. radiodurans cells that had been exposed to ionizing or UV radiation (15 kGy and 750 J/m2, respectively). The ringlike shape of the chromatin was maintained for 1 hour after exposure. However, as the post-irradiation incubation continued, the toroidal DNA structure underwent a transition into an open S-like morphology, followed by progressive spreading of DNA between two compartments through a membrane orifice. After 3 hours, this morphological reorganization culminated in the coalescence of two nucleoids (Fig. 1, E and F).

A recA-defective mutant of D. radiodurans(rec30), which is highly sensitive to both UV and ionizing radiation (13), also exhibited a toroidal DNA morphology that disappeared 90 min after exposure to UV or ionizing radiation. However, in contrast to wild-type cells, the recA mutant showed no evidence of DNA spreading between compartments or nucleoid fusion.

Partial compartmentalization and segregation of DNA without subsequent cell division are uncommon in bacteria. Even more unusual is the toroidal morphology adopted by the D. radioduransnucleoids. A similar structure has been observed only in metabolically inactive forms of life such as dormant spores and sperm cells (15). We suggest that these structural features play a crucial role in the radioresistance of D. radiodurans. The tight packaging and substantial order that characterize DNA toroids (15) render this structure into a relatively rigid matrix, in which DNA lateral continuity is maintained even when the DNA contains numerous breaks. Within this matrix, free DNA ends, generated by desiccation or irradiation, are kept firmly together because of restricted diffusion of DNA fragments.

We thus propose that the first phase of DNA repair in D. radiodurans involves template-independent yet error-free joining of DNA fragments within DNA toroids. Segregation of genome copies in different compartments is likely to further reduce the probability of errors by preventing the joining of DNA fragments from different chromosomes. This hypothesis is supported by two observations. First, in vitro studies have demonstrated that within DNA toroids, ligation of DNA fragments is markedly enhanced relative to the ligation rate in dispersed DNA structures (16, 17). Second, the chromatin in D. radiodurans retains substantially higher amounts of Mn2+ than the chromatin of other bacteria (18). Because Mn2+ is uniquely efficient in promoting toroidal DNA condensation upon dehydration (19, 20), these findings imply that D. radiodurans uses Mn2+ to regulate the extent of packaging and particular morphology of its genome copies.

To further assess the importance of DNA toroids, we followed up on the finding that exposure of D. radiodurans to high extracellular concentrations of Mn2+ (≥2.5 μM) substantially impairs its radioresistance without affecting its viability in unstressed conditions (21). We found that bacteria exposed to 100 μM Mn2+ appeared as aggregates of noncompartmentalized, fully separated bacteria, within which DNA toroids could no longer be detected, revealing instead a dispersed nucleoid structure (Fig. 2). Although the mechanism underlying these Mn2+-induced effects remains unclear, these observations indicate that the organism's DNA toroidal morphology is directly correlated with its DNA repair capacity.

Figure 2

Transmission electron micrographs of MnSO4-treated D. radiodurans. MnSO4(100 μM) was added to the growth medium at the late logarithmic phase (14 hours old), and the cells were incubated for an additional (A) 48 hours or (B) 6 days. Bacteria exposed to Mn2+ formed aggregates but were noncompartmentalized and had no detectable membrane orifices. (C to E) Different morphologies exhibited by DNA-stained cells (10) exposed to MnSO4 for 6 days, showing that in these cells, genomes, which are darkly stained, do not form toroids. BecauseD. radiodurans cells exposed to large extracellular concentrations of Mn2+ become radiation sensitive (21), the absence of DNA toroids indicates that toroidal packaging is correlated with radioresistance. Scale bars, 500 nm.

The repair model proposed here is consistent with biochemical data on D. radiodurans. An efficient RecA-independent DNA repair pathway was shown to be deployed in D. radiodurans immediately after DNA damage and to be followed by a RecA-dependent repair phase (13, 22). The initial mending pathway is interpreted in terms of template-independent joining of DNA fragments that substantially reduces the number of DNA fragments and thus facilitates a subsequent repair phase. Our observations imply that this second phase is promoted by fusion of nucleoids from two compartments (Fig. 1, E and F), which provides a platform for template-dependent recombination. The lack of nucleoid fusion in irradiated recA-deficient D. radiodurans mutants supports this premise, because these cells can carry out the RecA-independent DNA phase of repair but not the second, RecA-dependent phase (13). In addition, it has been argued that when bacteria contain several segregated copies of the genome, these copies would reveal different extents of packaging (23). Such differential packaging would explain how a single D. radiodurans cell maintains both dispersed chromosomes that allow metabolic activity (14) and DNA toroids that promote resistance.

The tight and ordered DNA packaging in D. radioduransmay facilitate DNA repair by promoting both template-independent DNA joining and RecA-dependent recombination. This packaging mode confers a constitutive state of readiness for and resistance to environmental stress that sets D. radiodurans apart from other bacteria.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5604/254/DC1

Materials and Methods

Fig. S1

References and Notes

  • * To whom correspondence should be addressed. E-mail: avi.minsky{at}weizmann.ac.il

REFERENCES AND NOTES

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