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Ordered Intracellular Reca–DNA Assemblies: a Potential Site of in Vivo Reca-Mediated Activities

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Ordered Intracellular Reca–DNA Assemblies: a Potential Site of in Vivo Reca-Mediated Activities Ordered intracellular RecA–DNA assemblies: A potential site of in vivo RecA-mediated activities Smadar Levin-Zaidman*, Daphna Frenkiel-Krispin*, Eyal Shimoni*, Ilana Sabanay*, Sharon G. Wolf†, and Abraham Minsky*‡ *Department of Organic Chemistry, and †Electron Microscopy Center, Weizmann Institute of Science, Rehovot 76100, Israel Edited by Charles M. Radding, Yale University School of Medicine, New Haven, CT, and approved March 9, 2000 (received for review December 8, 1999) The inducible SOS response increases the ability of bacteria to cope Materials and Methods with DNA damage through various DNA repair processes in which Electron Microscopy. Wild-type Escherichia coli AB1157 (recAϩ, the RecA protein plays a central role. Here we present the first lexAϩ) as well as various recA mutants were grown in LB medium study of the morphological aspects that accompany the SOS without NaCl to midlogarithmic phase. Cultures were then either response in Escherichia coli. We find that induction of the SOS treated with 100 ␮g of nalidixic acid or exposed to UV irradi- system in wild-type bacteria results in a fast and massive intracel- ation (20 J͞m2, 254 nm). After incubation at 37°C for 15, 30, 60, lular coaggregation of RecA and DNA into a lateral macroscopic 90, or 120 min, cells were fixed by ultrafast freezing in liquid assembly. The coaggregates comprise substantial portions of both ethane and cryosubstituted as described (7). Three independent the cellular RecA and the DNA complement. The structural features experiments were conducted for each time point, and in each of the coaggregates and their relation to in vitro RecA–DNA experiment, 800–1,000 cell slices were screened. In control networks, as well as morphological studies of strains carrying RecA experiments, chemical fixation was performed according to the mutants, are all consistent with the possibility that the intracellular RK-U procedure (8). Samples were embedded in Epon; thin assemblies represent a functional entity in which RecA-mediated sections were stained with 1% uranyl acetate and examined on DNA repair and protection activities occur. a Philips CM12 electron microscope operating at 100 kV. biocrystallization DNA repair homology search stress response ͉ ͉ ͉ In Situ Localization of DNA and RecA. Intracellular localization of DNA was performed with the DNA-specific stain osmium- ecA-mediated DNA recombination and repair processes ammine-SO2 (9). Grid-mounted thin sections of Epon- Rproceed through several sequential phases (1–3). A presyn- embedded bacteria were floated on 5 M HCl for 30 min at room aptic filament in which RecA molecules coat a single-stranded temperature, washed with distilled water, and treated with DNA substrate is initially formed. The filament acts then as a osmium ammine-B (Polysciences) in 8 M acetic acid͞40 mM sequence-specific DNA-binding entity, capable of searching and sodium metabisulfite for1hat37°C. Sections were then binding dsDNA sites that are homologous to the RecA-coated thoroughly rinsed with distilled water, dried, and studied without segment. Within the resulting joint species, DNA strand ex- additional staining. change and heteroduplex extension processes are promoted. The The intracellular distribution of RecA was determined by mechanism that enables a rapid search for DNA homology in immunogold labeling. Grid-mounted thin sections were incu- vivo, within a highly crowded and complex genome, remains bated for1honasaturated aqueous solution of sodium enigmatic. periodate and then treated with blocking solution (1% goat To reach its target, any sequence-specific DNA-binding pro- serum͞1% gelatin͞0.5% BSA in PBS) for 1 h. Grids were then tein must overcome two general obstacles: a minute cellular incubated for2hwithrabbit IgG anti-RecA polyclonal antibod- concentration of the target, and a vast excess of nontarget, yet ies (1:200 dilution with PBS). After a wash with the blocking still competitive, DNA sites. The search for a homologous DNA solution, grids were incubated with gold-conjugated goat anti- site conducted by the RecA–DNA presynaptic filament shares rabbit IgG for 1 h, dried, and stained with 2% uranyl acetate. All these hurdles, but is further encumbered by the uniquely adverse transactions were performed at room temperature. diffusion characteristics of its components. A DNA target corresponds to a segment that is part of, and embedded within, Image Reconstruction. Images of the intracellular crystals were the chromosome. This, and the large structural asymmetry of digitized with an Imacon Flextight scanner. Processing included DNA, conspire to minimize the diffusion constant of DNA sites crystal ‘‘unbending’’ and averaging with the MRC IMAGE software (4, 5). In a homology search executed by the RecA–DNA (10). The density maps were prepared with the CCP4 program MICROBIOLOGY filament, both the searching and the target entities are chromo- suite. somal DNA sites whose small diffusion constants drastically attenuate their encounter rate. How then does a RecA-mediated Results intracellular search evade the kinetic impediments that are Morphological Reorganization in DNA-Damaged Bacteria. Bacterial intrinsic to the nature of its components? chromatin is demarcated in electron micrographs of metaboli- Here we show that damages inflicted on bacterial DNA lead cally active cells as amorphous ribosome-free spaces that are to a rapid formation of an ordered intracellular assembly that irregularly spread over the cytoplasm (7, 8) (Figs. 1A and 2A). accommodates both RecA and DNA. We suggest that the striated morphology of this RecA–DNA assembly is capable of promoting an in vivo homology search by attenuating both the This paper was submitted directly (Track II) to the PNAS office. sampling volume and the dimensionality of the process. More- Abbreviations: dsDNA, double-stranded DNA. over, RecA was shown to protect chromosomal DNA from ‡To whom reprint requests should be addressed. E-mail: [email protected]. degradation (6) through unknown mechanisms. The tight crys- The publication costs of this article were defrayed in part by page charge payment. This talline packaging that is progressively assumed by the intracel- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. lular RecA–DNA assemblies as DNA damages accumulate is §1734 solely to indicate this fact. proposed to confer efficient DNA protection through physical Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.090532397. sequestration. Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.090532397 PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 ͉ 6791–6796 Downloaded by guest on October 3, 2021 Fig. 1. Electron microscopy of E. coli exposed to DNA-damaging agents. (A) Wild-type E. coli AB1157 at midlogarithmic phase. The dark particles are ribosomes. The ribosome-free spaces contain chromatin. (B) AB1157 cells treated with nalidixic acid for 30 min. A similar morphology is exhibited by UV-irradiated bacteria. (C) Cells treated with nalidixic acid in the presence of spermidine (2.5 mM) for 2 h. (D) High magnification of the assembly shown in C. Identical morphologies are detected in bacteria fixed by either cryo or chemical methods. Because these two fixation modes proceed through fundamentally different mechanisms, detection of the assemblies in both procedures indicates that these structures represent a genuine morphological feature. (Scale bars are 200 nm in A, B, and D, and 500 nm in C.) Wild-type E. coli cells exposed to DNA-damaging agents that should statistically be detected in a fifth of the very thin induce the SOS response reveal a strikingly different morphol- randomly cut cell slices, if present in all cells. Indeed, 25 Ϯ 5 ogy (Fig. 1B). In bacteria treated with nalidixic acid, which assemblies are found per 100 cells sections, indicating that they effects double-strand DNA breaks by stalling the activity of are formed in a vast majority of stressed bacteria. DNA gyrase (11), the DNA-containing ribosome-free regions Similar morphological traits, including the appearance of a can no longer be discerned. Instead, the ribosomes appear to be lateral assembly and the redistribution of ribosomes, are also uniformly distributed over the cytoplasm. The redistribution of detected shortly after exposure of wild-type bacteria to a UV ribosomes, and hence of chromatin, is accompanied by the pulse. Further unstressed incubation results in a rapid disap- appearance of a prominent array of parallel bundles. Continuous pearance of the assembly, indicating that the stress-induced treatment of the cells with nalidixic acid leads to a progressive effects are reversible. Significantly, UV irradiation causes DNA expansion of the assembly, whose average length increases from lesions in a replication-dependent pathway that is fundamentally 200 nm after 15 min to 800 Ϯ 100 nm after 60 min. Further different from the DNA-damaging mechanism of nalidixic acid incubation does not affect a significant expansion but results in (12). Thus, the massive intracellular reorganization represents a a higher lateral order, as indicated by the appearance of dif- generic outcome of SOS-inducing DNA lesions. fraction patterns. The average distance between bundles derived Positively charged polyamines promote the morphological from these patterns is 10 nm. At this stage, the lateral assembly modifications that are sustained by wild-type bacteria treated occupies a fifth of the cell volume, as deduced from measure- with DNA-damaging factors. Addition of spermidine to the ments on several hundred bacterial sections. Notably, when the growth media before the treatment with nalidixic acid or after dimension of the assemblies is a fifth of the cell volume, they UV irradiation accelerates the redistribution of ribosomes into 6792 ͉ www.pnas.org Levin-Zaidman et al. Downloaded by guest on October 3, 2021 MICROBIOLOGY Fig. 2. In situ localization of DNA and RecA. (A) Wild-type E. coli AB1157 cells at midlogarithmic phase, stained solely with the DNA-specific reagent osmium-ammine-SO2. The irregular spreading of chromatin over the cytoplasm is indicated.
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