Rad50 is not essential for the Mre11-dependent repair of DNA double-strand breaks in Halobacterium sp. strain NRC-1

Abstract

The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), and gamma radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.

FIG. 1.

Southern hybridization blots showing deletions of the mre11, rad50, and mre11-rad50 genes (A to G) and schematic of probe locations (H). Probes were designed to hybridize to the regions 500 bp immediately upstream of the mre11 coding region (mre11 probe I) (A), 500 bp immediately downstream of the rad50 coding region (rad50 probe I) (C and E), and within the coding regions of mre11 (mre11 probe II) (B and G) and rad50 (rad50 probe II) (D and F).

FIG. 2.

mRNA analysis for mre11, rad50, and mre11-rad50 genes in the background strain, deletion mutants, and wild-type Halobacterium. (A and B) RT-PCR analysis showing the presence or absence of mRNA transcripts in the AK071 background strain (lane 1), AK073 (Δrad50) (lane 2), AK072 (Δmre11) (lane 3), and wild-type Halobacterium (lane 4). A 500-bp fragment of the transcript from mre11 was identified in AK071, AK073, and the wild type but was lacking in AK072, as expected (A). A 500-bp fragment of the transcript from rad50 was identified in AK071, AK072, and the wild type but was lacking in AK073, as expected (B). (C) Northern hybridization blot showing the absence of mRNA transcription in AK073 (Δrad50) compared to the observed mRNA in AK071 (background strain). DNA probes were designed to hybridize to a 500-bp region within the rad50 transcript.

FIG. 3.

Generation time for strains AK071 (background), AK072 (Δmre11), AK073 (Δrad50), and AK074 (Δmre11Δrad50). Data shown are the averages of at least two replicates, with standard errors.

FIG. 4.

Survival of strains AK071 (background), AK072 (Δmre11), AK073 (Δrad50), and AK074 (Δmre11-Δrad50) after exposure to 50 μg/ml (black) and 100 μg/ml (gray) MNNG (A), 200 J/m² (black) and 350 J/m² (gray) UV-C radiation with recovery in the dark (B), γ radiation at doses of 2.5 kGy (black) and 5 kGy (gray) (C), and γ radiation at a dose of 2.5 kGy, followed by a 4-h incubation under standard culturing conditions and followed by a second dose of 2.5 kGy (D). Survival was calculated as the average ratio (N/No) of surviving CFU from treated (N) compared to untreated (No) cultures. Data shown are the averages of at least three replicates, with standard errors shown.

FIG. 5.

PFGE time course of recovery after exposure to 2.5 kGy of γ radiation. Samples were taken preirradiation (P) and immediately following irradiation (0) and every 4 h during the recovery up to 12 h and were embedded in InCert agarose plugs at a final density of 1 × 10⁹cells/ml. Plugs were digested with XbaI prior to gel electrophoresis. Images were taken from one of three independent replicates of this experiment.