DNA damage induces nucleoid compaction via the Mre11-Rad50 complex in the archaeon Haloferax volcanii

Abstract

In prokaryotes the genome is organized in a dynamic structure called the nucleoid, which is embedded in the cytoplasm. We show here that in the archaeon Haloferax volcanii, compaction and reorganization of the nucleoid is induced by stresses that damage the genome or interfere with its replication. The fraction of cells exhibiting nucleoid compaction was proportional to the dose of the DNA damaging agent, and results obtained in cells defective for nucleotide excision repair suggest that breakage of DNA strands triggers reorganization of the nucleoid. We observed that compaction depends on the Mre11-Rad50 complex, suggesting a link to DNA double-strand break repair. However, compaction was observed in a radA mutant, indicating that the role of Mre11-Rad50 in nucleoid reorganisation is independent of homologous recombination. We therefore propose that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate their targets, and facilitating the search for intact DNA sequences during homologous recombination.

Fig. 1

DNA damage and replication arrest induce nucleoid compaction. Fluorescence microscopy images of WT cells (H115) (phase contrast in blue, DNA in green) in (A) exponential phase, (B) and (C) after 1 h in presence of phleomycin or etoposide respectively, (D) and (E) 1 h after irradiation with doses of UV of 60 or 180 J m⁻² respectively and (F) after 1 h in presence of aphidicolin. G. Diverse morphologies of compacted nucleoids.

Fig. 2

H. volcanii responds to UV irradiation by nucleoid compaction. Mid-log phase WT cells (H26) were (A) stained directly with the nucleic acid stains acridine orange, Hoechst 33342, or propidium iodide, or (B) formaldehyde fixed and then ethanol permeabilized before staining. Results for both control and UV-treated (60 J m⁻²) cells are shown. The compaction effect is independent of the choice of stain and cell permeability.

Fig. 3

Nucleoid compaction depends on dose of DNA damage. A. Fluorescence microscopy images (phase contrast in blue, DNA in green) of WT (H115) cells before and after irradiation with 180 J m⁻² UV. B. Percentage of cells having a compacted nucleoid during the recovery after irradiation with 0, 60 or 180 J m⁻² UV as determined from microscopy images. Each point represents the mean (± SEM) values of at least three independent experiments. At least 300 cells were counted per time point per experiment. C. Fluorescence microscopy images of WT cells (H115) (phase contrast in blue, DNA in green) after irradiation with doses of 60 or 180 J m⁻² UV showing different degrees of nucleoid compaction. D. Relative area occupied by the compacted nucleoid inside cells during the recovery after irradiation with doses of 60 or 180 J m⁻² UV. At least 121 individual cells were analysed; the asterisk corresponds to P < 0.05 by unpaired Student's t-test.

Fig. 4

Nucleoid compaction detected by flow cytometry. A. Flow cytometry analysis of cell size versus acridine orange fluorescence intensity after irradiation of WT H. volcanii (H115) with a dose of 180 J m⁻² UV. At 1 h after UV irradiation, the three populations (A, B and C) described in the results are highlighted. B. Percentage of population C among populations C and B during the recovery, after irradiation with doses of UV of 0, 60 or 180 J m⁻². Each point represents the mean (± SEM) values of at least four independent experiments.

Fig. 5

Nucleoid compaction depends on Mre11-Rad50 but not homologous recombination. A. Percentage of cells with a compacted nucleoid determined by flow cytometry in WT (H115) and mre11 rad50 (m-r) mutant (H204) after irradiation with 0, 60 and 180 J m⁻² UV. Each point represents the mean (± SEM) values from at least three independent experiments. B. Relative area occupied by the compacted nucleoid inside WT (H115) and mre11 rad50 (m-r) (H204) cells after irradiation with 60 or 180 J m⁻² UV. At least 121 individual cells had been analysed, the asterisk corresponds to P < 0.05 by unpaired Student's t-test. C. Represents the percentage of cells with a compacted nucleoid determined by flow cytometry in WT (H115) or ΔradA mutant (H607) after irradiation with doses of UV of 0, 60 or 180 J m⁻². D. Fluorescence microscopy images (phase contrast in blue, DNA in green) of ΔradA cells (H607) 3 h after irradiation with 180 J m⁻² UV.

Fig. 6

Actinomycin D inhibition of nucleoid compaction. A. Fluorescence microscopy image (phase contrast in blue, DNA in green) of WT cells (H115) after irradiation with 180 J m⁻² UV in the presence of DMSO or actinomycin D. B. Percentage of WT cells (H115) with a compacted nucleoid determined by flow cytometry after irradiation with 0 or 180 J m⁻² UV in the presence of DMSO, anisomycin or actinomycin D. Each point represents the mean (± SEM) values from three independent experiments. C. Percentage of WT (H115) and mre11 rad50 (m-r) mutant (H204) cell with a compacted nucleoid determined by flow cytometry after irradiation with 0 and 180 J m⁻² UV in the presence of DMSO or actinomycin D. Each point represents the mean (± SEM) values from three independent experiments.

Fig. 7

Role of nucleotide excision repair in nucleoid compaction. (A) and (B) represent the percentage of cells with a compacted nucleoid determined by flow cytometry in WT (H26) and ΔuvrA mutant (H509) after irradiation with 0, 60 or 180 J m⁻² UV. Each point represents the mean (± SEM) values from four independent experiments.