Rad50 is dispensable for the maintenance and viability of postmitotic tissues

Abstract

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.

FIG. 1.

Rad50-inducible construction and deletion. (A) The germ line region targeted for conditional deletion is shown as Rad50. The targeting vector was constructed to contain LoxP sites and a puromycin resistance (Puro^r) marker flanking exons 1 and 2. Conventional gene targeting methods were used to generate embryonic stem cells harboring the conditional allele (Rad50^ind). Expression of Cre recombinase in cells or mice harboring this allele results in generation of the Rad50⁻ allele. (B) Integration of the targeting construct was confirmed via Southern blotting using BamHI- and XbaI-digested genomic DNA with 3′- and 5′-specific probes, respectively (depicted as black bars in panel A). (C) Genotype PCR strategies were devised to differentiate the Rad50 loci. Primers F2 and R1 (see panel A for primer diagram) amplify Rad50 (or Rad50^Δ), a larger fragment from Rad50^ind, and no product from Rad50⁻. Primers F1 and R1 amplify a Rad50⁻-specific band. WT, wild type.

FIG. 2.

Rad50 deletion in cultured cells results in genomic instability. (A) Rad50 levels from lentivirus-Cre-infected SV40-transformed ear fibroblasts were detected by Western blotting. Actin is shown as a loading control. (B) The frequency of metaphase aberrations was determined for control (Rad50^+/ind) and Rad50-deficient (Rad50^Δ/ind) cells, as indicated on the graph, in uninfected cells (−) and cells at 4 and 6 days postdeletion. (C) Colony formation was assessed in control cells (white bars), inducible cells transfected with a vector expressing Rad50 cDNA (gray bars), and Rad50-deficient cells (red bars).

FIG. 3.

Rad50-deficient cells do not exhibit telomere defects. (A) Telomere FISH-stained metaphase spreads were analyzed for the presence of fused chromosomes. Fusions with and without telomere sequence at junctions were tabulated. An example of a fusion lacking telomere sequence is shown from a Rad50^Δ/− spread. (B) Spontaneous and IR-induced 53BP1 foci in Rad50 control (white bars), Rad50-deficient (gray bars), and Trf2-deficient (black bars) cells were analyzed for colocalization with telomeres. Values represent percentages of 53BP1 foci exhibiting TIFs (*, IR-treated Trf2 deficient cells were not analyzed). Examples of 53BP1 (red)- and TelC (green)-stained cells from Rad50-deficient (C and D) and Trf2-deficient (E and F) cultures.

FIG. 4.

Rad50 deletion in bone marrow, quiescent liver, and postmitotic Purkinje cells. (A) Ear, liver, and bone marrow from adult Rad50^+/ind and Rad50^Δ/ind Mx-Cre⁺ mice were analyzed for deletion of the Rad50^ind allele at 7 weeks after pI-pC injection; kidney and liver from pI-pC-injected Rad50^Δ/ind Mx-Cre⁺ mice were analyzed before (−) and 12 days after PH. (B) Immunohistochemical staining of cerebellar sections from Pcp2-Cre⁺ control (Rad50^+/Δ) and mutant (Rad50^Δ/−) mice with Rad50 antibody. Loss of nuclear Rad50 staining in the Purkinje cell layer (PCL) of mutant mice is evident (arrows). (C) Gaits were recorded at 4 and 16 months, and average stride lengths and widths were calculated for control and Rad50^Δ/− Pcp2-Cre⁺ mice.

FIG. 5.

Liver regeneration is normal in Rad50-deficient cells, but hepatocytes exhibit indices of DNA damage. (A) Kidney and liver sections from control and Rad50^Δ/ind Mx-Cre⁺ mice were immunohistochemically stained with Rad50 antibody. Examples from mice sacrificed 4 weeks after pI-pC injection and 6 days post-PH are shown. (B) Percent liver weight recovery was calculated, and results were graphed for control and mutant mice up to 8 days post-PH. (C) Immunohistochemical staining of liver sections prepared from control and mutant mice were used to determine the percent γ-H2AX-positive hepatocytes before and after PH. Examples of γ-H2AX-labeled sections from Rad50^+/− and Rad50^Δ/− livers at 2 days post-PH are shown. quiesc, quiescent.

FIG. 6.

Rad50-deficient hepatocytes divide at a reduced rate. The mitotic index at 2 days post-PH was determined by immunofluorescent labeling with phospho-H3 and DAPI staining of sections from control and Rad50^Δ/− livers (A). (B) Phospho-H3-positive cells at 2 days post-PH were classified according to the indicated stages, and the percentages in each class were graphed for control and mutant mice. Representative images from the various stages are shown. Red, phospho-H3 (Ph-H3); pseudo-green, DAPI.